Physiology of the aging bone and mechanisms of action of bisphosphonates

Fragility fractures, a major public health concern, are expected to further increase due to aging of the world populations because age remains a cardinal, independent determinant of fracture risk. With aging the balance between bone formation and resorption during the remodeling process becomes negative, with increased resorption and reduced formation. Bisphosphonates (BPs) are widely prescribed anti-resorptive agents that inhibit osteoclasts attachment to bone matrix and enhance osteoclast apoptosis. BPs can be divided into nitrogen-containing (N-BPs) and non-nitrogen-containing BPs (non-N-BPs). Both classes induce apoptosis but they evoke it differently. Several studies have examined the molecular mechanisms underlying BPs’ effects on osteoclasts and bone remodeling. N-BPs (alendronate, risedronate, zoledronate) inhibit the intracellular mevalonate pathway and protein isoprenylation, via the enzyme farnesyl pyrophosphate synthase. N-BPs act by competition, binding to the natural substrate-binding site of the enzyme. The less potent non-N-BPs (etidronate, clodronate), do not inhibit the mevalonate pathway and protein isoprenylation, but are metabolized intracellularly to metabolites, which are cytotoxic analogs of ATP. N-BPs represent the first choice treatment for diseases associated with excessive bone resorption, such as fragility fractures (due to postmenopausal-, male, glucocorticoid- and transplant-induced osteoporosis), Paget’s disease of bone, and bone metastasis. Better understanding of BPs’ effects on osteoblasts/osteocytes (e.g., preventing apoptosis) and differential distribution may further help explain anti-fracture benefit and bone quality effects. Lower affinity BPs (e.g., risedronate) may allow better access to osteocyte network. Effects of BPs on bone senescence, cancer cells apoptosis and prevention of cardiovascular calcifications may open new avenues for biogerontological research.     

The Molecular Mechanisms of Action of Bisphosphonates

BPs can be grouped into two general classes according to their chemical structure and the molecular mechanism by which they inhibit osteoclast-mediated bone resorption. The simple BPs can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulate intracellularly in osteoclasts, causing osteoclast cell death by apoptosis. By contrast, the more potent N-BPs inhibit FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids that are required for the prenylation of small GTPase signalling proteins necessary for osteoclast function. Inhibition of FPP synthase in cells other than osteoclasts also appears to account for the adverse effects of N-BPs in vivo (including the acute phase reaction) and for the anti-tumour effects of N-BPs in vitro.     

Bisphosphonate mechanism of action

The nitrogen-containing bisphosphonates (N-BPs), alendronate and risedronate, are the only pharmacologic agents shown to prevent spine and nonvertebral fractures associated with postmenopausal and glucocorticoid-induced osteoporosis. At the tissue level, this is achieved through osteoclast inhibition, which leads to reduced bone turnover, increased bone mass, and improved mineralization. The molecular targets of bisphosphonates (BPs) have recently been identified. This review will discuss the mechanism of action of BPs, focusing on alendronate and risedronate, which are the two agents most widely studied. They act on the cholesterol biosynthesis pathway enzyme, farnesyl diphosphate synthase. By inhibiting this enzyme in the osteoclast, they interfere with geranylgeranylation (attachment of the lipid to regulatory proteins), which causes osteoclast inactivation. This mechanism is responsible for N-BP suppression of osteoclastic bone resorption and reduction of bone turnover, which leads to fracture prevention.     

A novel third generation bisphosphonate, minodronate (YM529), prevented proliferation and migration of hepatocellular carcinoma cells through inhibition of mevalonate pathway

Aim:  Skeletal metastases and bone metasitasis are a common occurrence in patients with advanced hepatocellular carcinoma (HCC). Bisphosphonates (BPs), which are used for the treatment of osteoporosis and tumor-associated hypercalcemia, have recently been reported to decrease skeletal morbidity in patients with metastatic bone disease. Several studies revealed that nitrogen-containing BPs (N-BPs) could inhibit tumor growth and migration, indicating the possibility that N-BPs have direct inhibitory effects. We aimed to determine the effects of novel a N-BP (YM529) on human HCC cells in vitro .
Methods:  HCC cells were treated with various concentrations of YM529 and the growth inhibition rate was determined. Apoptosis was evaluated by caspase-3/7 assay and caspase-9 cleavage detection. The effects of YM529 on the migration of HCC cells induced by hepatocyte growth factor (HGF) were determined by cell migration assay. To evaluate the involvement of the mevalonate pathway, farnesol (FOH) and geranylgeraniol (GGOH) were added.
Results:  YM529 inhibited the proliferation of HCC cells in a dose-dependent manner. The activation of caspase-3/7 and cleavage of caspase-9 demonstrated the involvement of apoptosis in cytotoxicity. GGOH reduced the growth inhibitory effect of YM529 and suppressed the induction of caspase-3/7 activities by YM529 on HCC cells. YM529 inhibited tumor cell migration induced by HGF and this effect was reduced by co-treatment with GGOH.
Conclusion:  YM529 inhibited the cell proliferation and migration of HCC cells, implicating the involvement of the mevalonate pathway. These results suggest that N-BPs are potential agents for the treatment of HCC skeletal metastases.     

In vivo effects of bisphosphonates on the osteoclast mevalonate pathway

Estrogen deficiency is a leading cause of osteoporosis associated with increased osteoclastic bone resorption. In vitro studies indicate that the clinically used nitrogen-containing bisphosphonates (N-BPs) such as alendronate (ALN), risedronate (RIS) and ibandronate (IBA) suppress bone resorption via inhibition of the mevalonate pathway enzyme farnesyl diphosphate (FPP) synthase in osteoclasts (Ocs). The object of this study was to test the hypothesis that N-BPs inhibit the mevalonate pathway of Ocs in vivo. The mevalonate pathway enzyme hydroxymethyl-glutaryl-coenzyme A reductase (HMGR), is modulated by feedback inhibition from downstream metabolites. We therefore evaluated the in vivo expression of HMGR in Ocs from animals treated with BP. The N-BPs, ALN, IBA and RIS, selectively suppressed HMGR expression in up to 85% of rat tibia osteoclasts, after 48 hr treatment. Etidronate and clodronate, bisphosphonates that do not inhibit FPP synthase, were without effect. Simvastatin treatment opposed ALN reduction of HMGR expression, suggesting regulation by a metabolite(s) between mevalonate and FPP. These data provide the first in vivo evidence for N-BP effects on the mevalonate pathway in osteoclasts, and strongly support the hypothesis that N-BPs act via this mechanism.     

Effect of glucocorticoids on the biologic activities of myeloma cells: inhibition of interleukin-1 beta osteoclast activating factor-induced bone resorption

Regulatory effects of glucocorticoids (dexamethasone) on myeloma cells as well as bone resorption in multiple myeloma were investigated. Glucocorticoids significantly inhibited proliferation of myeloma cells, and decreased the messenger RNA (mRNA) expressions of interleukin-6 (IL-6) and secretory type immunoglobulin G (IgG). The inhibitory effects of glucocorticoids on myeloma cell proliferation could be due to the decreased expression of IL-6 mRNA, decreased IL-6 production, and thus suppression of autocrine growth by IL-6, which is an autocrine growth factor for myeloma cells as reported previously (Nature 332:83, 1988). Glucocorticoids also inhibited M-protein secretion by decreasing the levels of secretory type Ig mRNA. On the other hand, because IL-1 beta rather than lymphotoxin is considered to be a major osteoclast activating factor (OAF) produced by myeloma cells, and glucocorticoids decreased the expression of IL-1 beta mRNA and markedly suppressed the bone resorbing activity induced by IL-1 beta OAF in 45Ca-release bone resorption assay, it is suggestive that glucocorticoids could inhibit bone resorption induced by IL-1 beta OAF in multiple myeloma. Therefore, from these data it is concluded that glucocorticoids could be more effective chemotherapeutic agents in multiple myeloma than we expected, especially with regards to the inhibitory effects on proliferation and M-protein secretion from myeloma cells, as well as bone resorption by myeloma cells.     

SDX-308, a nonsteroidal anti-inflammatory agent, inhibits NF-kappaB activity, resulting in strong inhibition of osteoclast formation/activity and multiple myeloma cell growth

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities, additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac, known as SDX-308, and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101, a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation, and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308, as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model, RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity, potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study.     

Differential effects of glucocorticoid on recruitment and activity of osteoclasts induced by normal and osteocalcin-deficient bone implanted in rats.

Prolonged glucocorticoid excess is associated with bone loss. Among the contributory factors are glucocorticoids' suppression of bone formation and stimulation of bone resorption. In this study, the effects of glucocorticoids on bone resorption were evaluated in a rodent model. Subcutaneous implants of devitalized mineralized bone particles (BPs) elicit the recruitment of progenitor cells and their differentiation to osteoclasts which resorb the BPs. The effects of glucocorticoids on both the recruitment and the activity of cells induced by normal BPs were distinguished based upon when treatment was initiated. When treatment with hydrocortisone or dexamethasone was initiated at the time of BP implantation, the recruitment of bone-resorbing cells was impaired and a subsequent decrease in BP resorption was found. On the other hand, when treatment was initiated on day 7, glucocorticoids increased osteoclastic resorption and tartrate-resistant acid phosphatase activity. We also tested hydrocortisone's effect to stimulate the activity of cells associated with osteocalcin-deficient BPs. As previously reported, BPs deficient in osteocalcin were poorly resorbed as a result of decreased formation and activity of osteoclasts. Hydrocortisone had an even more pronounced effect in stimulating the low level resorption of the osteocalcin-deficient BP implants than of the normal BP implants. These findings show differential effects of glucocorticoids on two aspects of bone resorption: they inhibit the recruitment and/or differentiation of bone-resorbing cells, but they stimulate the activity of existing osteoclastic cells. The ability of glucocorticoids to increase resorption of normal bone and to overcome resistance to resorption of osteocalcin-deficient bone suggests an important regulatory effect of glucocorticoids in the activation of osteoclasts to increase bone resorption.     

Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone

Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.     

The molecular mechanism of action of the antiresorptive and antiinflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis

OBJECTIVE: The primary aims of this study were to determine whether clodronate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(beta,gamma-dichloromethylene) triphosphate (AppCCl2p) by osteoclasts and macrophages in vivo, and to determine whether intracellular accumulation of this metabolite accounts for the antiresorptive and antimacrophage effects of clodronate. To compare the mechanism of action of clodronate and alendronate, effects on protein prenylation in osteoclasts and macrophages in vivo were also assessed. METHODS: High-performance liquid chroma-tography-mass spectrometry was used to determine whether rabbit osteoclasts (purified ex vivo with immunomagnetic beads) metabolize clodronate, and whether rat peritoneal macrophages metabolize liposome-encapsulated clodronate, following in vivo administration. The effects of clodronate and AppCCl2p on bone resorption, osteoclast number, and apoptosis in vitro were compared. Using an antibody to the unprenylated form of RaplA, effects on protein prenylation were assessed by Western blot analysis of osteoclast and peritoneal macrophage lysates from bisphosphonate-treated animals. RESULTS: AppCCl2p could be detected in extracts from osteoclasts purified from clodronate-treated rabbits. Intracellular accumulation of AppCCl2p caused a reduction in the number of osteoclasts, increased osteoclast apoptosis, and inhibited bone resorption in vitro. These effects were indistinguishable from those of clodronate. Liposome-encapsulated clodronate was also metabolized to AppCCl2p by rat peritoneal macrophages in vivo. Liposome-encapsulated clodronate caused an increase in peritoneal macrophage apoptosis in ex vivo cultures that was indistinguishable from the increase in apoptosis caused by liposome-encapsulated AppCCl2p. Unlike alendronate, clodronate and its metabolite did not affect prenylation of the small GTPase RaplA in osteoclasts or macrophages in vivo. CONCLUSION: These results provide the first direct evidence that the antiinflammatory and antiresorptive effects of clodronate on macrophages and osteoclasts in vivo occur via the intracellular formation of AppCCl2p.     

Bisphosphonates: the molecular targets and mechanisms of action

Bisphosphonates directly act on osteoclasts to inhibit bone resorption and used most widely to treat osteoporosis. The compounds can be classified into two groups with different modes of action. Nitrogen containing bisphosphonates exert their effects by inhibiting a key enzyme in the mevalonate pathway. The specific target is the isoprenoid biosynthetic enzyme, farnesyl pyrophosphate synthase, which is indispensable for protein prenylation and activation of intracellular signalling proteins, including the small GTPases Rho, Rac, Cdc42 and Ras. The disruption of the function of these key enzymes may explain the loss of osteoclast activity and induction of apoptosis. Whereas the first generation of bisphosphonates such as etidronate and clodronate (nitrogen deficient bisphosphonates) can be incorporated into nonhydrolysable analogues of ATP that may inhibit ATP-dependent intracellular events. Bisphosphonates are highly effective to inhibit bone resorption and increase bone mineral density, although their precise mechanisms of molecular action are not completely understood.     

Breast cancer cells interact with osteoblasts to support osteoclast formation.

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption. Recently the osteoclast differentiation factor (ODF), better termed RANKL (receptor activator of NF-kappaB ligand), expressed by osteoblasts has been cloned as well as its cognate signaling receptor, receptor activator of NFkappaB (RANK), and a secreted decoy receptor osteoprotegerin (OPG) that limits RANKL's biological action. We determined that the breast cancer cell lines MDA-MB-231, MCF-7, and T47D as well as primary breast cancers do not express RANKL but express OPG and RANK. MCF-7, MDA-MB-231, and T47D cells did not act as surrogate osteoblasts to support osteoclast formation in coculture experiments, a result consistent with the fact that they do not express RANKL. When MCF-7 cells overexpressing PTH-related protein (PTHrP) were added to cocultures of murine osteoblasts and hematopoietic cells, osteoclast formation resulted without the addition of any osteotropic agents; cocultures with MCF-7 or MCF-7 cells transfected with pcDNAIneo required exogenous agents for osteoclast formation. When MCF-7 cells overexpressing PTHrP were cultured with murine osteoblasts, osteoblastic RANKL messenger RNA (mRNA) levels were enhanced and osteoblastic OPG mRNA levels diminished; MCF-7 parental cells had no effect on RANKL or OPG mRNA levels when cultured with osteoblastic cells. Using a murine model of breast cancer metastasis to bone, we established that MCF-7 cells that overexpress PTHrP caused significantly more bone metastases, which were associated with increased osteoclast formation, elevated plasma PTHrP concentrations and hypercalcaemia compared with parental or empty vector controls.     

The synthetic triterpenoid TP-222 inhibits RANKL stimulation of osteoclastogenesis and matrix metalloproteinase-9 expression

OBJECTIVE: Receptor activator of nuclear factor-kappaB ligand (RANKL) promotes osteoclast differentiation from monocyte precursors by inducing a cohort of genes, including tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). A family of synthetic triterpenoids with antiinflammatory and pro-apoptotic properties was described to modulate differentiation in monocytic cell lineages. We therefore investigated the ability of the potent and bioavailable synthetic triterpenoid TP-222 to inhibit RANKL-induced osteoclast formation and MMP-9 expression from monocytic precursor cells. METHODS: Osteoclast formation was assayed by staining for TRAP-positive multinucleated cells. MMP-9 expression was measured by quantitative RT-PCR, Western blot, immunohistochemistry, and gel zymography. In vivo effects of TP-222 were assessed by daily intraperitoneal injection of 4-week-old mice for 7 days followed by measurement of osteoclast number and MMP-9 expression at the cartilage/bone junction of the epiphyseal growth plate. RESULTS: RANKL promoted and TP-222 (300 nM) inhibited osteoclast formation in cultures of RAW264.7 cells or bone marrow-derived monocytes. RANKL also induced MMP-9 expression in RAW264.7 cells and this was reduced by concurrent or subsequent addition of TP-222. TP-222 treatment significantly reduced the mean number of osteoclasts present at the cartilage/bone interface compared to vehicle-injected control mice. Morphometric analyses of tissue sections showed that TP-222 treatment reduced the amount of immunoreactive MMP-9 present in both mononucleated pre-osteoclasts and osteoclasts. CONCLUSION: Our data demonstrate that TP-222 inhibits osteoclast formation and MMP-9 expression in vitro and in vivo, and suggest that triterpenoids may be useful compounds for modulating bone resorption diseases.     

Bisphosphonates as potential adjuvants for patients with cancers of the digestive system

Best known for their anti-resorptive activity in bone, bisphosphonates(BPs) have generated interest as potential antineoplastic agents given their pleiotropic biological effects which include antiproliferative, antiangiogenic and immune-modulating properties. Clinical studies in multiple malignancies suggest that BPs may be active in the prevention or treatment of cancer. Digestive tract malignancies represent a large and heterogeneous disease group, and the activity of BPs in these cancers has not been extensively studied. Recent data showing that some BPs inhibit human epidermal growth factor receptor(HER) signaling highlight a potential therapeutic opportunity in digestive cancers, many of which have alterations in the HER axis. Herein, we review the available evidence providing a rationale for the repurposing of BPs as a therapeutic adjunct in the treatment of digestive malignancies, especially in HER-driven subgroups.     

Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase.

We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NO-generating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption.     

Epidermal growth factor receptor tyrosine kinase inhibitors and bone metastases: different mechanisms of action for a novel therapeutic application?

The paper by Angelucci et al. published in the current issue of Endocrine-Related Cancer suggests a potential, novel application of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in the treatment of bone metastases. Interestingly, activity of anti-EGFR agents on the pathogenesis and progression of bone metastases has been described in previous reports, and a number of different mechanisms seem to be involved in this phenomenon. Anti-EGFR agents have a direct activity on tumour cells in which they produce growth inhibition, apoptosis, and reduced invasive capacity through the inhibition of molecules associated with tissue invasion such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9. In addition, these compounds have an anti-angiogenic activity, either direct by affecting the proliferation and survival of endothelial cells, or indirect by blocking the production of vascular endothelial growth factor (VEGF) in bone marrow stromal cells and in tumour cells. Finally, EGFR-TKIs can inhibit recruitment of osteoclasts in bone lesions, by affecting the ability of bone marrow stromal cells to induce osteoclast differentiation and activation. Taken together, these findings strongly support prospective clinical trials of anti-EGFR agents in cancer patients with bone metastases in order to define their role in the management of bone disease.     

Myeloma bone disease

Bone destruction is a hallmark of multiple myeloma, and recent studies demonstrated a strong interdependence between tumor progression and bone resorption. Increased bone resorption as a major characteristic of multiple myeloma is caused by osteoclast activation and osteoblast inhibition (uncoupling). Myeloma cells alter the local regulation of bone metabolism by increasing the receptor activator of NF-kappaB ligand (RANKL) and decreasing osteoprotegerin (OPG) expression within the bone marrow microenvironment, thereby stimulating the central pathway for osteoclast formation and activation. In addition, they produce the chemokines MIP-1alpha, MIP-1beta and SDF-1alpha, which also increase osteoclast activity. Furthermore, myeloma cells suppress osteoblast function by the secretion of osteoblast inhibiting factors, e.g. Dickkopf (DKK)-1. The resulting bone destruction releases several cytokines, which in turn promote myeloma cell growth. Therefore, the inhibition of bone resorption could stop this vicious circle and not only decrease myeloma bone disease, but also the tumor progression. Preclinical studies provided strong evidence that the suppression of the osteoclast activity using bisphosphonates, RANKL blockade or inhibition of MIP-1alpha or MIP-1beta is effective both in reducing myeloma bone disease and tumor growth and therefore may offer an important treatment strategy in multiple myeloma.     

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.     

A carboxyl-terminal peptide from the parathyroid hormone-related protein inhibits bone resorption by osteoclasts

PTH-related protein (PTHrP) interacts, via its amino-terminal 34 residues, with PTH receptors on osteoblasts to stimulate osteoclastic bone resorption indirectly. We now report that mature hPTHrP-(1-141) (EC50, approximately 10(-11) M) and a carboxyl-terminal fragment, PTHrP-(107-139) (EC50, approximately 10(-15) M), are potent inhibitors of resorption in an isolated rat osteoclast bone resorption assay, whereas hPTHrP-(1-108) and hPTHrP-(1-34) are inactive in this respect. PTHrP-(107-139) also inhibits resorption in a rat long bone organ culture system and reduces osteoclast spreading. PTHrP-(107-139) does not act on osteoclasts via a cAMP signal transduction mechanism, but its effects may be mediated by protein kinase-C. This previously unrecognized action of PTHrP, to inhibit osteoclastic bone resorption directly, indicates that PTHrP may be a precursor of multiple biologically active peptides with differing physiological functions.     

Endothelin inhibits osteoclastic bone resorption by a direct effect on cell motility: implications for the vascular control of bone resorption.

The abundance of endothelin (ET)-producing endothelial cells in bone marrow and the proximity of these cells to bone-resorbing osteoclasts prompted us to evaluate the action of ET-1 on osteoclast function. Osteoclasts disaggregated from neonatal rat long bones were settled onto devitalized cortical bone substrate, and resorption was quantified by morphometry. The supernatant tartrate-resistant acid phosphatase activity was determined by a spectrophotometric method using paranitrophenol phosphate as substrate. Cell motility was quantified by time lapse video- and computer-assisted image processing using an empirical procedure for morphometric analysis. Cytosolic free calcium levels ([Ca2+]i) were measured in single cells by an indo 1-based microspectrofluorimetric method. Using the area of bone resorbed per slice as response, we found that ET-1 caused a significant (P = 0.011) concentration-dependent inhibition of osteoclastic bone resorption (EC50 = 2.5 nM) without inhibiting acid phosphatase secretion. Exposure of isolated osteoclasts to ET-1 also led to a marked concentration-dependent inhibition of osteoclast motility (EC50 = 7.9 nM; P = 0.013; t1/2 = 18 min) without significant effects on cell spread area. These effects of ET-1 were reversible after removing the peptide, and the cells remained viable during the experiments. In addition, ET-1 did not elevate [Ca2+]i at the concentrations tested. The results suggest that ET-1 specifically interacts with an osteoclast receptor to inhibit osteoclastic bone resorption and cell motility. As the concentration of ET-1 required for osteoclast inhibition was similar to that reported for smooth muscle contraction, it is possible that ET-1, produced locally from the bone marrow endothelial cell, might play a primary role in osteoclast regulation.