Over 80% of women with advanced breast cancer ultimately develop bone metastases which account significantly for morbidity and mortality. Breast cancer metastases in bone can cause intractable pain, bone fracture, spinal cord compression and hypercalcaemia. It also signifies that the malignant process is incurable since, once tumour cells become lodged in the skeleton, therapy can only be given with palliative intent. This includes analgesics, radiation therapy and systemic treatments such as hormone or chemotherapy. The events leading to the development of bone lesions in patients with carcinoma of the breast are poorly understood. However, histomorphometric studies have shown that tumour cells are adjacent to actively resorbing osteoclasts (Boyde et al, 1986) and it has been suggested that breast carcinoma cells possess the capacity to recruit and stimulate osteoclasts by producing stimulatory factors (Boyde et al, 1986). Parathyroid hormone related peptide (PTHrP) is thought to be a major candidate factor produced by breast cancer cells which may promote osteoclastic activity at metastatic sites in bone (Powell et al, 1991).Bisphosphonates (BPs) are analogues of endogenous pyrophosphates in which a carbon atom replaces the central atom of oxygen. In vivo, bisphosphonates bind strongly to hydroxyapatite on the bone surface and are preferentially delivered to sites of increased bone formation or resorption. They are potent inhibitors of osteoclast-mediated bone resorption (Boonecamp et al, 1986) and are effective in lowering serum calcium concentrations in patients with hypercalcaemia of malignancy (Ryzen et al, 1985;Kanis et al, 1987). Bisphosphonates are also used in the treatment of Paget's disease of bone (Altman et al, 1973;Plasmans et al, 1978) and bone lesions associated with multiple myeloma (Berenson et al, 1996). The mechanisms by which bisphosphonates inhibit osteoclast-mediated bone resorption remain to be determined, but may involve inhibition of formation of osteoclasts from immature precursor cells (Boonecamp et al, 1986;Lowik et al, 1988;Hughes et al, 1989) and/or direct inhibition of resorption via induction of apoptosis in mature osteoclasts (Lowik et al, 1988;Hughes et al, 1995;Selander et al, 1996). More recently, several reports have indicated that bisphosphonates have direct effects on other cell types which may have important implications in the treatment of patients with cancer-induced bone disease. Treatment with intravenous pamidronate (Hortobagyi et al, 1998) and oral clodronate (Paterson et al, 1993) have been reported to reduce the frequency of skeletal complications in established bone disease. Oral clodronate has also been shown to decrease the frequency of skeletal metastases in women who had recurrent breast cancer but without bony involvement (Kanis et al, 1996). Moreover, oral clodronate has recently been shown to increase survival in women with breast cancer, when given at the time of initial diagnosis to patients who have bone marrow micrometastases at the time of surgery fo...

Reports implicating Wnt signalling in the regulation of bone mass have prompted widespread interest in the use of Wnt mimetics for the treatment of skeletal disorders. To date much of this work has focused on their anabolic effects acting on cells of the osteoblast lineage. In this study we provide evidence that Wnts also regulate osteoclast formation and bone resorption, through a mechanism involving transcriptional repression of the gene encoding the osteoclastogenic cytokine receptor activator of NFκB ligand (RANKL or TNFSF11) expressed by osteoblasts. In co-cultures of mouse mononuclear spleen cells and osteoblasts, inhibition of GSK3β with LiCl or exposure to Wnt3a inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells compared with controls. However, these treatments had no consistent effect on the differentiation, survival or activity of osteoclasts generated in the absence of supporting stromal cells. Activation of Wnt signalling downregulated RANKL mRNA and protein expression, and overexpression of fulllength β-catenin, but not transcriptionally inactive β-catenin ΔC(695-781), inhibited RANKL promoter activity. Since previous studies have demonstrated an absence of resorptive phenotype in mice lacking LRP5, we determined expression of a second Wnt co-receptor LRP6 in human osteoblasts, CD14+ osteoclast progenitors and mature osteoclasts. LRP5 expression was undetectable in CD14-enriched cells and mature human osteoclasts, although LRP6 was expressed at high levels by these cells. Our evidence of Wnt-dependent regulation of osteoclastogenesis adds to the growing complexity of Wnt signalling mechanisms that are now known to influence skeletal function and highlights the requirement to develop novel therapeutics that differentially target anabolic and catabolic Wnt effects in bone.

The importance of the vascular supply for bone is well-known to orthopaedists but is still rather overlooked within the wider field of skeletal research. Blood supplies oxygen, nutrients and regulatory factors to tissues, as well as removing metabolic waste products such as carbon dioxide and acid. Bone receives up to about 10% of cardiac output, and this blood supply permits a much higher degree of cellularity, remodelling and repair than is possible in cartilage, which is avascular. The blood supply to bone is delivered to the endosteal cavity by nutrient arteries, then flows through marrow sinusoids before exiting via numerous small vessels that ramify through the cortex. The marrow cavity affords a range of vascular niches that are thought to regulate the growth and differentiation of hematopoietic and stromal cells, in part via gradients of oxygen tension. The quality of vascular supply to bone tends to decline with age and may be compromised in common pathological settings, including diabetes, anaemias, chronic airway diseases and immobility, as well as by tumours. Reductions in vascular supply are associated with bone loss. This may be due in part to the direct effects of hypoxia, which blocks osteoblast function and bone formation but causes reciprocal increases in osteoclastogenesis and bone resorption. Common regulatory factors such as parathyroid hormone or nitrates, both of which are potent vasodilators, might exert their osteogenic effects on bone via the vasculature. These observations suggest that the bone vasculature will be a fruitful area for future research.

Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7-and 13-day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21-fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short-term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3-day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast-mediated calcium release, an effect equivalent to that of prostaglandin E 2 (PGE 2 ), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously-overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.

The skeletons of land vertebrates contain a massive reserve of alkaline mineral (hydroxyapatite), which is ultimately available to buffer metabolic H+ if acid-base balance is not maintained within narrow limits. The negative impact of acidosis on the skeleton has long been known but was thought to result from passive, physicochemical dissolution of bone mineral. This brief, selective review summarizes what is now known of the direct functional responses of bone cells to extracellular pH. We discovered that bone resorption by cultured osteoclasts is stimulated directly by acid. The stimulatory effect is near-maximal at pH 7.0, whereas above pH 7.4, resorption is switched off. In bone organ cultures, H+-stimulated bone mineral release is almost entirely osteoclast-mediated, with a negligible physicochemical component. Acidification is the key requirement for osteoclasts to excavate resorption pits in all species studied to date, and extracellular H+ may thus be regarded as the long-sought osteoclast activation factor. Acid-activated osteoclasts can be stimulated further by agents such as parathyroid hormone, 1,25-dihydroxycholecalciferol, and receptor activator of nuclear factor kappaB ligand. Osteoclasts may respond to pH changes via H+-sensing ion channels such as transient receptor potential vanilloid 1, a nociceptor that is also activated by capsaicin. Acidosis also exerts a powerful, reciprocal inhibitory effect on the mineralization of bone matrix by cultured osteoblasts. This is caused by increased hydroxyapatite solubility at low pH, together with selective inhibition of alkaline phosphatase, which is required for mineralization. Diets or drugs that shift acid-base balance in the alkaline direction may provide useful treatments for bone loss disorders.