αvβ3 Integrin antagonists as inhibitors of bone resorption

The _vβ₃ integrin is a non-covalent, heterodimeric, cell-surface protein that is expressed with varying density on numerous cell types, including osteoclasts, vascular smooth muscle cells, endothelial cells and a variety of tumour cells. Functionally, _vβ₃ mediates a diverse range of biological events including the adhesion of osteoclasts to bone matrix, smooth muscle cell migration and angiogenesis. Specifically, there has been significant attention focused on the preparation of inhibitors of _vβ₃ for use as inhibitors of bone resorption, in recognition of the medical need for improved prevention and treatment of osteoporosis. Herein, we summarise the pertinent chemistry and biological advances in the medicinal design and biological evaluation of peptide and small molecule _vβ₃ antagonists as inhibitors of bone resorption.     

Nonpeptide alpha(v)beta(3) antagonists. 1. Transformation of a potent, integrin-selective alpha(IIb)beta(3) antagonist into a potent alpha(v)beta(3) antagonist

Modification of the potent fibrinogen receptor (alpha(IIb)beta(3)) antagonist 1 generated compounds with high affinity for the vitronectin receptor alpha(v)beta(3). Sequential modification of the basic N-terminus of 1 led to the identification of the 5,6,7, 8-tetrahydro[1,8]naphthyridine moiety (THN) as a lipophilic, moderately basic N-terminus that provides molecules with excellent potency and selectivity for the integrin receptor alpha(v)beta(3). The THN-containing analogue 5 is a potent inhibitor of bone resorption in vitro and in vivo. In addition, the identification of a novel, nonpeptide radioligand with high affinity to alpha(v)beta(3) is also reported.     

Alpha v integrin affinity/specificity and antiangiogenesis effect of a novel tetraaza cyclic peptide derivative, SU015, in various species

The present study was undertaken to define the alpha v beta3 and alpha v beta5 binding potency and specificity of SU015, an integrin antagonist. SU015 inhibited alpha v beta3-mediated human umbilical vein endothelial cell or 293/beta3-transfected CHO cell adhesion to fibrinogen, with IC50 values of 0.21 +/- 0.11 muM and 0.32 +/- 0.02 microM. SU015 demonstrated comparable affinity to alpha v beta5 as compared with alpha v beta3 affinity, as well as a relatively high degree of specificity for human alpha v beta3- and alpha v beta5-mediated functions, as compared with other human integrins, including alphaIIbbeta3 (IC50 >100 microM), alpha5/beta1 (IC50 >100 microM), and alpha4/beta1 (IC50 >100 microM). SU015 demonstrated different degrees of species specificity in blocking alpha v beta3-mediated cellular adhesion, with relatively higher affinity to monkey (IC50 = 0.10 microM) and dog (IC50 = 1.30 microM) endothelial or smooth muscle cell alpha v beta3-mediated adhesion. Additionally, SU015 demonstrated a high degree of alpha v beta3 and alpha v beta5 specificity as compared with alpha4beta1-, alpha5beta1-, or alpha IIb beta3-mediated binding in the above species. In conclusion, SU015 is an alpha v beta3 and alpha v beta5 antagonist with relatively higher potency and specificity as compared with alpha IIb beta3, alpha5beta1, or alpha4beta1 integrins. Additionally, comparable alpha v beta3 and alpha v beta5 affinity for SU015 was demonstrated with human and monkey endothelial cells. These data also suggest that this bicyclic RGD analogue linked to a linker at the bottom leaves the RGD at the top available for binding and allows for conjugation with radioisotope for imaging and radiotherapy.     

Nanomolar small molecule inhibitors for alphav(beta)6, alphav(beta)5, and alphav(beta)3 integrins

Integrin adhesion receptors frequently recognize a core amino acid sequence, Arg-Gly-Asp, in their target ligands. Inhibitors with the ability to inhibit one or a small subset of such RGD-dependent integrins have been invaluable in defining their biological function. Here, we have characterized low molecular weight inhibitors for their ability to specifically inhibit alphav(beta)6 integrin, a fibronectin/tenascin receptor. As of yet, no nonpeptidic inhibitor of alphav(beta)6 was known. New peptidomimetic and nonpeptidic compounds were examined in isolated integrin binding assays and in cell adhesion assays for their ability to block alphav(beta)6, alphav(beta)3, alphav(beta)5, and alphalIb(beta)3 integrins. The compounds are based on an aromatically substituted beta amino acid or glutaric acid derivative as an acidic center and an aminopyridyl or guanidyl residue as a basic mimetic. We found several classes of inhibitors with different selectivities, especially mono- or biselectivity on the alpha(v)-integrins alphav(beta)6 and alphav(beta)3, and nanomolar activity. Furthermore, nearly all compounds are inactive on alphaIIb(beta)3. Compound 11 is the first specific, peptidomimetic inhibitor of the alphav(beta)6 integrin receptor.     

双醋瑞因对破骨细胞性骨破坏的抑制作用及机制

目的考察双醋瑞因对破骨细胞生成及骨破坏功能是否具有抑制作用，以及双醋瑞因抑制破骨细胞的作用是否与影响成骨细胞中OPG及RANKL的表达有关。方法MC3T3-E1细胞与骨髓前体细胞共培养生成破骨细胞，将TRAP染色阳性、细胞核数目≥3个的细胞作为破骨细胞，计数生成的破骨细胞。计数IL-1β作用前后典型的骨吸收陷窝以观察破骨细胞的活性。应用Western blotting法、流式细胞术及RT-PCR法在蛋白水平及基因水平观察MC3T3-E1细胞中RANKL及OPG的表达。结果双醋瑞因可显著抑制IL-1β作用下破骨细胞的生成及其骨陷窝形成功能，sRANKL的加入可逆转双醋瑞因的上述作用。双醋瑞因可在基因及蛋白水平上调MC3T3-T1细胞中OPG/RANKL的比例。结论双醋瑞因具有抑制IL-1β诱导的破骨细胞性骨破坏的作用，这一作用可能与其抑制MC3T3-E1细胞中RANKL表达同时促进OPG表达有关。     

Osteoclasts resorb protein-free mineral (Osteologic discs) efficiently in the absence of osteopontin

Osteopontin (OPN) is both a matrix protein in mineralized tissues and a cytokine, and it has a pivotal role in osteoclast-mediated bone resorption. Here, using a proprietary hydroxyapatite substitute for bone mineral (Osteologic discs), we investigated the requirement for OPN in mineral resorption. Resorption pits formed in the Osteologic discs, revealed by staining with silver nitrite (Von Kossa stain), were analyzed using the NIH Image J program, which can determine the number of pits formed per unit area, their average size, and the fractional area resorbed. After a preincubation of bone marrow cells from OPN -/- and OPN +/+ mice with M-CSF to allow the multiplication of osteoclast precursors on cell culture plastic, osteoclast formation on both Osteologic discs and standard cell culture plates was induced with soluble receptor activator of NFkappaB ligand, sRANKL. We did not detect a dramatic difference in osteoclast formation between OPN +/+ and OPN -/- cells, as judged by staining for tartrate-resistant acid phosphatase in osteoclasts formed on cell culture plastic, nor was there a significant difference in the ability of the osteoclasts to form resorption pits in the Osteologic discs. Additionally, none of six different anti-OPN monoclonal antibodies had a significant and reproducible effect on the formation or subsequent functioning of the OPN+/+ osteoclasts. These studies suggest that, in contrast to what has been found for normal bone, the efficiency of dissolution of a ceramic, protein-free (excepting protein adsorbed from the culture medium) hydroxyapatite/tri-calcium phosphate substrate by osteoclasts is not substantially enhanced by endogenous or exogenous OPN.     

Blocking of sodium and potassium ion-dependent adenosine triphosphatase-α1 with ouabain and vanadate suppresses cell-cell fusion during RANKL-mediated osteoclastogenesis

To examine the possible enrolment of Na(+)/K(+)-ATPase during osteoclast differentiation, Na(+)/K(+)-ATPase inhibitors, including ouabain and vanadate, were used in this study. These inhibitors significantly inhibited cell-cell fusion of RAW264.7 cells and bone marrow cells induced by RANKL. Interestingly, in response to RANKL-stimulation, ouabain and vanadate decreased the number of large TRAP+ osteoclasts in the culture of RAW264.7 cells, as well as bone marrow cells. In contrast, the number of small TRAP+ osteoclasts either increased in RAW264.7 cells or were otherwise less affected in bone marrow cells than large TRAP+ osteoclasts. Large TRAP+ osteoclasts are defined as having ≥ 10 nuclei/cell and having more potency in bone resorption than small multinuclear osteoclasts with <9 nuclei/cell. Na(+)/K(+)-ATPase α1 and β2 mRNAs were detected in sRANKL-stimulated RAW264.7 cells. Moreover, real-time quantitative PCR showed that ouabain and vanadate suppressed the RANKL-dependent induction of the osteoclast fusion-promotion molecule DC-STAMP at the mRNA level. Finally, and importantly, RNAi-mediated suppression of Na(+)/K(+)-ATPase α1 resulted in a diminished number of large TRAP+ osteoclasts in the sRANKL-stimulated RAW264.7 cells, along with the decreased level of DC-STAMP mRNA expression. These findings strongly suggest that blockage of the Na(+)/K(+)-ATPase α1 subunit by ouabain or vanadate caused the inhibition of RANKL-induced cell-cell fusion, resulting in the generation of large osteoclasts through suppression of DC-STAMP expression. Thus, in addition to its known function of sodium and potassium ion exchange during bone resorption by mature osteoclasts, this study has revealed a novel molecular role of the Na(+)/K(+)-ATPase α1 subunit in osteoclastogenesis.     

Molecular model of the alpha(IIb)beta(3) integrin

A molecular model of the alpha(IIb)beta(3) integrin has been developed utilizing (i). the crystal structure of alpha(v)beta(3), (ii). homology model of the alpha(IIb) subdomain, and (iii). the docking of alpha(IIb)beta(3)/alpha(v)beta(3) dual and selective inhibitors into the putative binding sites of alpha(IIb)beta(3) and alpha(v)beta(3). Since the binding sites of these integrins are located at the interface between the two heads of the individual subunits, only the alpha(IIb)beta(3) head region is modeled. The 3D conformations of two loops in alpha(IIb), whose residues have been implicated in non-peptide ligand binding, could not be determined from homology with alpha(v) alone. Mutagenesis data and the modeling of small ligand binding contributed to the rational design of these loop conformations. The final energy minimized loop conformations exhibit permissible phi/psi angles and contribute to a binding site model of alpha(IIb)beta(3) that is consistent with both the known mutagenesis studies and in-house structure-activity relationships. The charged residues alpha(IIb):E117 and beta(3):R214 are found to dominate the ligand-protein binding interaction. The previously identified "exosite" is also identified as a hydrogen bond, hydrophobic or pi-pi interaction with Y190, similar to the recently proposed binding model of alpha(v)beta(3).     

Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro

The effects of an important new anti-inflammatory agent, the selective cyclooxygenase-2 inhibitor celecoxib, on bone resorption and osteoclastogenesis elicited by the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), the endotoxin lipopolysaccharide (LPS), and the systemic hormones 1alpha,25-dihydroxyvitamin D(3) and parathyroid hormone were examined in vitro. Bone resorption was evaluated by measuring calcium released into the culture medium in a neonatal mouse calvarial bone organ culture. Osteoclastogenesis was evaluated by measuring tartrate-resistant acid phosphatase activity in the cells in cocultures of bone marrow cells and osteoblastic cells and in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. Celecoxib (0.1 microM) completely inhibited the calcium release induced by IL-1beta, TNF-alpha, and LPS. The resorptive effect of 1alpha,25-dihydroxyvitamin D(3) was inhibited partially by celecoxib. In contrast, celecoxib did not inhibit the calcium release elicited by parathyroid hormone or prostaglandin E(2). Celecoxib (0.1 microM) also markedly inhibited osteoclastogenesis induced by these stimulators of bone resorption except for PGE(2) in the coculture system, whereas it failed to inhibit osteoclastogenesis in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. These results indicate that, under certain conditions, cyclooxygenase-2-dependent prostaglandin synthesis is critical for the bone resorption induced by IL-1beta, TNF-alpha, and LPS, and for the osteoclastogenesis induced by these pro-inflammatory molecules and calciotropic hormones. The prevention of prostaglandin synthesis by inflammatory cytokines in bone cells could contribute to the efficacy of celecoxib in preventing bone loss in rheumatoid arthritis.     

alpha(v)beta(3) Integrin-mediated drug resistance in human laryngeal carcinoma cells is caused by glutathione-dependent elimination of drug-induced reactive oxidative species

As a model for determination of the role of integrins in drug resistance, we used alpha(v)beta(3) integrin-negative human laryngeal carcinoma cell line (HEp2) and three HEp2-derived cell clones with a gradual increase of alpha(v)beta(3) integrin expression. The alpha(v)beta(3) integrin expression protects cells from cisplatin, mitomycin C, and doxorubicin. In HEp2-alpha(v)beta(3) integrin-expressing cells, the constitutive expression of Bcl-2 protein and the level of glutathione (GSH) were increased compared with HEp2 cells. Pretreatment of HEp2-alpha(v)beta(3) integrin-expressing cells with an inhibitor of GSH synthesis, buthionine sulfoximine (BSO), decreased the level of GSH and partially reverted drug resistance to all above-mentioned drugs, but it did not influence the expression of Bcl-2. Sensitivity to selected anticancer drugs did not change with overexpression of Bcl-2 in HEp2 cells, nor with silencing of Bcl-2 in HEp2-alpha(v)beta(3) integrin-expressing cells, indicating that Bcl-2 is not involved in resistance mechanism. There was no difference in DNA platination between HEp2 and HEp2-alpha(v)beta(3) integrin-expressing cells, indicating that the mechanism of drug resistance is independent of cisplatin detoxification by GSH. A strong increase of reactive oxidative species (ROS) formation during cisplatin or doxorubicin treatment in HEp2 cells was reduced in HEp2-alpha(v)beta(3) integrin-expressing cells. Since this increased elimination of ROS could be reverted by GSH depletion, we concluded that multidrug resistance is the consequence of GSH-dependent increased ability of alpha(v)beta(3)-expressing cells to eliminate drug-induced ROS.     

Current topics in pharmacological research on bone metabolism: inhibitory effects of bisphosphonates on the differentiation and activity of osteoclasts

Despite the extensive use of bisphosphonates (BPs) in the treatment of metabolic bone diseases associated with increased osteoclastic bone resorption, the precise mechanism of their action on bone metabolism is still unclear. To clarify at which stages of osteoclast differentiation and activation that BPs influence, we examined the osteoclasts generated from mononuclear precursors and osteoclasts in the calvaria by laser scanning confocal microscopy. The studies showed that BPs inhibit lipopolysaccharide- or parathyroid hormone-induced osteoclast differentiation, fusion, attachment, actin ring formation, and activation and that both beta3 integrin and osteopontin have an important role in cytoskeletal rearrangements associated with cell attachment and resorption in osteoclasts.     

Quercetin suppresses bone resorption by inhibiting the differentiation and activation of osteoclasts

Although quercetin has suppressed bone resorption in several animal studies, its target cells and the mechanism of its action related to bone resorption has not been fully elucidated. We investigated the effect of quercetin on the differentiation and activation of osteoclasts. We used cocultures of mouse spleen cells and ST2 cells, and cultures of osteoclast progenitor cells [M-CSF-dependent (MD) cells from mouse bone marrow and murine monocytic RAW 264 (RAW) cells]. Quercetin dose-dependently inhibited osteoclast-like (OCL) cell formation at 2-5 microM concentration in both the coculture and MD cell culture. Quercetin inhibited the increase of tartrate-resistant acid phosphatase (TRAP) activity of mononuclear preosteoclasts (pOCs) induced by receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) in both MD and RAW cell cultures. Quercetin reversely induced the disruption of actin rings in OCLs. Quercetin also suppressed both pit formation induced by osteoclasts on dentine slices and PTH-stimulated (45)Ca release in mouse long bone cultures. These results suggest that osteoclast progenitors as well as mature osteoclasts, are quercetin's target cells in relation to bone resorption, and that quercetin's suppressive effect on bone resorption results from both its inhibitory effect on the differentiation of osteoclast progenitor cells into pOCs and from its disruptive effect on actin rings in mature osteoclasts.     

Isoxazolines as potent antagonists of the integrin alpha(v)beta(3)

Starting with lead compound 2, we sought to increase the selectivity for alpha(v)beta(3)-mediated cell adhesion by examining the effects of structural changes in both the guanidine mimetic and the substituent alpha to the carboxylate. To prepare some of the desired aminoimidazoles, a novel reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found that guanidine mimetics with a wide range of pK(a)'s were potent antagonists of alpha(v)beta(3). In general, it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity for alpha(v)beta(3)-mediated adhesion versus alpha(IIb)beta(3)-mediated platelet aggregation, with selectivity of approximately 3 orders of magnitude observed for compounds 3g and 3h. It was also found in this series that the alpha-substituent was required for potent activity and that 2,6-disubstituted arylsulfonamides were optimal. In addition, the selective alpha(v)beta(3) antagonist 3h was found to be a potent inhibitor of alpha(v)beta(3)-mediated cell migration.     

Pharmacological characterization of recombinant N-type calcium channel (Cav2.2) mediated calcium mobilization using FLIPR

The N-type voltage-gated calcium channel (Ca(v)2.2) functions in neurons to regulate neurotransmitter release. It comprises a clinically relevant target for chronic pain. We have validated a calcium mobilization approach to assessing Ca(v)2.2 pharmacology in two stable Ca(v)2.2 cell lines: alpha1(B), alpha2delta, beta(3)-HEK-293 and alpha1(B), beta(3)-HEK-293. Ca(v)2.2 channels were opened by addition of KCl and Ca(2+) mobilization was measured by Fluo-4 fluorescence on a fluorescence imaging plate reader (FLIPR(96)). Ca(v)2.2 expression and biophysics were confirmed by patch-clamp electrophysiology (EP). Both cell lines responded to KCl with adequate signal-to-background. Signals from both cell lines were inhibited by omega-conotoxin (ctx)-MVIIa and omega-conotoxin (ctx)-GVIa with IC(50) values of 1.8 and 1nM, respectively, for the three-subunit stable, and 0.9 and 0.6nM, respectively, for the two-subunit stable. Other known Ca(v)2.2 blockers were characterized including cadmium, flunarizine, fluspirilene, and mibefradil. IC(50) values correlated with literature EP-derived values. Novel Ca(v)2.2 pharmacology was identified in classes of compounds with other primary pharmacological activities, including Na(+) channel inhibitors and antidepressants. Novel Na(+) channel compounds with high potency at Ca(v)2.2 were identified in the phenoxyphenyl pyridine, phenoxyphenyl pyrazole, and other classes. The highest potency at Ca(v)2.2 tricyclic antidepressant identified was desipramine.     

Binding model for nonpeptide antagonists of alpha(v)beta(3) integrin

A binding model for nonpeptide antagonists of integrin alpha(v)beta(3) has been developed through docking analyses utilizing the MMFFs force field and the recently published crystal structure, 1JV2. Results of this docking study have led to the identification of a novel binding model for selective antagonists of alpha(v)beta(3) over alpha(IIb)beta(3) integrins. Four different chemical classes are shown to bind in a similar fashion providing a measure of confidence in the proposed model. All alpha(v)beta(3) and alpha(IIb)beta(3) antagonists have a basic nitrogen separated some distance from a carboxylic acid to mimic RGD. For the alpha(v)beta(3) antagonists under present consideration, these charged ends are separated by twelve bonds. The basic nitrogen of the active alpha(v)beta(3) ligands are shown to interact with D150 of alpha(v) and the ligands' carboxylic acid interact with R214 of beta(3) while adopting an extended conformation with minimal protein induced internal strain. In addition, an energetically favorable interaction is found with all of the active alpha(v)beta(3) molecules with Y178 of alpha(v) when docked to the crystallographically determined structure. This novel interaction may be characterized as pi-pi stacking for the most active of the alpha(v)beta(3) selective antagonists. The proposed model is consistent with observed activity as well as mutagenicity and photoaffinity cross-linking studies of the alpha(v)beta(3) integrin.     

Mitogenic effects of cytokines on smooth muscle are critically dependent on protein kinase A and are unmasked by steroids and cyclooxygenase inhibitors

Excessive smooth muscle growth occurs within the context of inflammation associated with certain vascular and airway diseases. The inflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) have been shown previously to inhibit mitogen-stimulated smooth muscle growth through a mechanism presumed to be dependent on the induction of cyclooxygenase-2, prostaglandins, and activation of the cAMP-dependent protein kinase (PKA). Using both molecular and pharmacological strategies, we demonstrate that the mitogenic effects of IL-1beta and TNF-alpha on cultured human airway smooth muscle (ASM) cells are tightly regulated by PKA activity. Suppression of induced PKA activity by either corticosteroids or cyclooxygenase inhibitors converts the cytokines from inhibitors to enhancers of mitogen-stimulated ASM growth, and biological variability in the capacity to activate PKA influences the modulatory effect of cytokines. Promitogenic effects of IL-1beta are associated with delayed increases in p42/p44 and phosphoinositide-3 kinase activities, suggesting a role for induced autocrine factors. These findings suggest a mechanism by which mainstream therapies such as corticosteroids or cyclooxygenase inhibitors could fail to address or exacerbate the pathogenic smooth muscle growth that occurs in obstructive airway and cardiovascular diseases.     

骨质疏松症破骨细胞的形成与骨吸收活性的研究

目的 分析骨质疏松症骨髓单核细胞向破骨细胞转化及其骨吸收活性的变化。了解骨质疏松骨量丢失的发生机理。方法 分离、诱导培养骨质疏松症骨髓单核细胞，观察细胞的生长与分化状况，测定培养早期的细胞分泌细胞因子的水平，以抗酒石酸酸性磷酸酶（TRAP）染色和噬骨试验检测破骨细胞的形成率及其骨吸收活性，结果 在地塞米松和维生素-D3（1，25-OH2D3）的诱导下，骨髓单核细胞可向多核巨细胞转化，并呈现TRAP阳性反应及明显的噬骨性，骨质疏松症组单核细胞早期可高水平分泌白细胞介素1（IL-1β），白细胞介素6（IL-6），随之发生的TRAP阳性细胞转化率和骨吸收活性高于非骨质疏松对照组。结论 骨髓单核细胞向破骨细胞转化及其骨吸收活性的增强，是骨质疏松骨量丢失的原因之一，因单核细胞异常高水平产生的IL-1β和IL-6在其中起介导作用。     

Integrin alpha v beta 3 antagonists for anti-angiogenic cancer treatment

Direct contact between cellular and extracellular matrix (ECM) proteins is necessary for a diverse array of physiologic processes including cellular activation, migration, proliferation, and differentiation. These direct interactions are modulated by cell adhesion molecules (CAMs) such as integrins, selectins, cadherins, and immunoglobulins. Integrin signaling also plays a key role in tumor growth, angiogenesis, and metastasis. Recent advances in the discovery and characterization of CAMs and their receptors, most notably integrin alpha(v)beta(3), and the clarification of their roles in disease states have laid the groundwork for the development and clinical implementation of novel anti-cancer treatments. Integrin alpha (v)beta(3) is a glycoprotein membrane receptor which recognizes ECM proteins expressing an arginine-glycine-aspartic acid (RGD) peptide sequence. The receptor is highly expressed on activated tumor endothelial cells, but not resting endothelial cells and normal organ systems, thus making alpha(v)beta(3) an appropriate target for anti-angiogenic therapeutics. In addition, alpha(v)beta(3) is also expressed on tumor cells, allowing for both tumor cell and tumor vasculature targeting of anti-integrin therapy. Throughout the past decade, numerous patents have been published and issued using alpha(v)beta(3) antagonists for the prevention and/or treatment of cancer, with many antagonists demonstrating positive pre-clinical anti-angiogenic and anti-tumor results. This review will focus on the key points and distinguishing factors for patents which use antibodies, RGD peptides, non-RGD peptides, peptidomimetics, and amine salts as alpha(v)beta(3) antagonists.     

Fluoride decreased osteoclastic bone resorption through the inhibition of NFATc1 gene expression

Over the past two decades, fluoride effects on osteoclasts have been evaluated; however, its molecular mechanisms remain unclear. In this study, we investigated the effect of fluoride on osteoclast formation, function, and regulation using osteoclasts formed from mice bone marrow macrophages treated with the receptor activator of NF‐κB ligand and macrophage colony‐stimulating factor. Our data showed that fluoride levels ≤ 8 mg/L had no effect on osteoclast formation; however, it significantly reduced osteoclast resorption at 0.5 mg/L. Fluoride activity on bone resorption occurred through the inhibition of nuclear factor of active T cells (NFAT) c1 expression. Furthermore, the expression of its downstream genes, including the dendritic cell‐specific transmembrane protein, c‐Src, the d2 isoform of vacuolar (H+) ATPase v0 domain, matrix metalloproteinase 9, and cathepsin K were decreased, leading to impaired osteoclast acidification, reduced secretion of proteolytic enzymes, and decreased bone resorption. In summary, our results suggested that fluoride has different roles in osteoclast formation and function. Fluoride ≤ 8 mg/L did not impact osteoclast formation; however, it significantly decreased the resorption activity of newly formed osteoclasts. The molecular mechanism of fluoride action may involve inhibition of NFATc1 and its downstream genes. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 588–595, 2014.     

Novel anti-cancer strategy in bone tumors by targeting molecular and cellular modulators of bone resorption

Tumor cells alter the balanced process of bone formation and bone resorption mediated respectively by osteoblasts and osteoclasts, leading to the disruption of the normal equilibrium and resulting in a spectrum of osteolytic to osteoblastic lesions. This review will summarize research on molecules that play direct and essential roles in the differentiation and activity of osteoclasts, and the role of these molecules in bone destruction caused by cancer. Results from experimental models suggest that the Receptor Activator of NF-kB Ligand (RANKL), a member of the TNF superfamily is a common effector of bony lesions in osteolysis caused by primary and secondary bone tumors. Therefore, osteoclast represents an attractive target across a broad range of tumors that develop in bone. Elucidation of the mechanisms of RANKL interactions with its activator (RANK) and decoy (osteoprotegerin: OPG) receptors has enable the development of pharmacological inhibitors of RANKL (and of its signalling pathway) which have been recently patented, with potential for the treatment of cancer-induced bone disease. Blocking bone resorption by specific other drugs such as bisphosphonates, inhibitors of cathepsin K (the main enzyme involved in bone resorption mechanisms) or signalling pathways regulating osteoclast differentiation and activation is also a promising target for the treatment of osteolysis associated to bone tumors.