Upregulation of endogenous farnesyl diphosphate synthase overcomes the inhibitory effect of bisphosphonate on protein prenylation in Hela cells.

Nitrogen-containing bisphosphonates (N-BPs) such as zoledronic acid (ZOL) are the gold standard treatment for diseases of excessive bone resorption. N-BPs inactivate osteoclasts via inhibition of farnesyl diphosphate synthase (FPPS), thereby preventing the prenylation of essential small GTPases. Not all patients respond to N-BP therapy to the same extent, and some patients, for example with tumour-associated bone disease or Paget's disease, appear to develop resistance to N-BPs.

Osteoporosis - a current view of pharmacological prevention and treatment.

Postmenopausal osteoporosis is the most common bone disease, associated with low bone mineral density (BMD) and pathological fractures which lead to significant morbidity. It is defined clinically by a BMD of 2.5 standard deviations or more below the young female adult mean (T-score =-2.

RANK-dependent autosomal recessive osteopetrosis: characterization of five new cases with novel mutations.

Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder attributed to reduced bone resorption by osteoclasts. Most human AROs are classified as osteoclast rich, but recently two subsets of osteoclast-poor ARO have been recognized as caused by defects in either TNFSF11 or TNFRSF11A genes, coding the RANKL and RANK proteins, respectively. The RANKL/RANK axis drives osteoclast differentiation and also plays a role in the immune system.

Transfection of osteoclasts and osteoclast precursors.

Osteoclasts and their precursors have traditionally been considered difficult cells to transfect using standard approaches. Here, we describe several methods for transfection of mature osteoclasts and their precursors using the Amaxa™ Nucleofector system, lentiviruses, and adenoviruses.

Isolation and purification of rabbit osteoclasts.

Newborn rabbits provide a useful and readily available source of authentic mature osteoclasts, which can be easily isolated directly from the long bones in relatively large numbers, compared to other rodents. Primary cultures of authentic rabbit osteoclasts on resorbable substrates in vitro are an ideal model of osteoclast behaviour in vivo, and for some studies may be preferable to osteoclast-like cells generated in vitro from bone marrow cultures or from human peripheral blood, for example in assessing osteoclast-mediated bone resorption independently of effects on osteoclast formation. Rabbits also provide a particularly useful model for determining the effects of pharmacological agents on osteoclasts in vivo, by isolating osteoclasts using immunomagnetic bead separation (with an antibody to α(V)β(3)) at the desired time following in vivo administration of the drug.

Absence of glutamine supplementation prevents differentiation of murine calvarial osteoblasts to a mineralizing phenotype.

Osteoblasts in vitro differentiate from a proliferating to a mineralizing phenotype upon transfer to a medium rich in beta-glycerophosphate and ascorbic acid. The nutritional requirements of the cells at different stages of this differentiation process are not known. In other cell types, nutritional supplementation during surgery can improve the outcome in terms of speed of patient recovery and prognosis.

The skeleton: a multi-functional complex organ: the role of key signalling pathways in osteoclast differentiation and in bone resorption.

Osteoclasts are the specialised cells that resorb bone matrix and are important both for the growth and shaping of bones throughout development as well as during the process of bone remodelling that occurs throughout life to maintain a healthy skeleton. Osteoclast formation, function and survival are tightly regulated by a network of signalling pathways, many of which have been identified through the study of rare monogenic diseases, knockout mouse models and animal strains carrying naturally occurring mutations in key molecules. In this review, we describe the processes of osteoclast formation, activation and function and discuss the major transcription factors and signalling pathways (including those that control the cytoskeletal rearrangements) that are important at each stage.

Signal peptide mutations in RANK prevent downstream activation of NF-κB.

Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF-κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF-κB occurred in HEK293 cells overexpressing wild-type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene.

Biochemical and molecular mechanisms of action of bisphosphonates.

This review describes the key discoveries over the last 15 years that have led to a clearer understanding of the molecular mechanisms by which bisphosphonate drugs inhibit bone resorption. Once released from bone mineral surfaces during bone resorption, these agents accumulate intracellularly in osteoclasts. Simple bisphosphonates such as clodronate are incorporated into non-hydrolysable analogues of adenosine triphosphate, which induce osteoclast apoptosis.

Increase in NF-kappaB binding affinity of the variant C allele of the toll-like receptor 9 -1237T/C polymorphism is associated with Helicobacter pylori-induced gastric disease.

Colonization of the gastric mucosa by Helicobacter pylori can lead to serious clinical outcomes, including gastric cancer. Toll-like receptors (TLRs) play an important role in the host response to H. pylori through the recognition of pathogen-associated molecular patterns.

Zoledronic acid induces formation of a pro-apoptotic ATP analogue and isopentenyl pyrophosphate in osteoclasts in vivo and in MCF-7 cells in vitro.

Background And Purpose: Bisphosphonates (BPs) are highly effective inhibitors of bone resorption. Nitrogen-containing bisphosphonates (N-BPs), such as zoledronic acid, induce the formation of a novel ATP analogue (1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester triphosphoric acid; ApppI), as a consequence of the inhibition of farnesyl pyrophosphate synthase and the accumulation of isopentenyl pyrophosphate (IPP). ApppI induces apoptosis, as do comparable metabolites of non-nitrogen-containing bisphosphonates (non-N-BPs).

Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations.

Autosomal-Recessive Osteopetrosis (ARO) comprises a heterogeneous group of bone diseases for which mutations in five genes are known as causative. Most ARO are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (also called RANKL or TRANCE, coding for the RANKL protein), a master gene driving osteoclast differentiation along the RANKL-RANK axis. RANKL and RANK (coded for by the TNFRSF11A gene) also play a role in the immune system, which raises the possibility that defects in this pathway might cause osteopetrosis with immunodeficiency.

A comparison between the effects of hydrophobic and hydrophilic statins on osteoclast function in vitro and ovariectomy-induced bone loss in vivo.

Statins potently inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, blocking downstream biosynthesis of isoprenoid lipids and causing inhibition of protein prenylation. Prenylated signaling molecules are essential for osteoclast function, consistent with our previous observation that mevastatin can inhibit osteoclast activity in vitro. Several reports suggest that statins may also have an anabolic effect on bone and stimulate osteoblast differentiation.

The matricellular protein CYR61 inhibits osteoclastogenesis by a mechanism independent of alphavbeta3 and alphavbeta5.

Cysteine-rich protein 61 (CYR61/CCN1) belongs to the family of CCN matricellular proteins. Most of the known effects of CCN proteins appear to be due to binding to extracellular growth factors or integrins, including alpha(v)beta(3) and alpha(v)beta(5). Although CYR61 can stimulate osteoblast differentiation, until now the effect of CYR61 on osteoclasts was unknown.

Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis.

Bisphosphonates such as alendronate and zoledronate are blockbuster drugs used to inhibit osteoclast-mediated bone resorption. Although the molecular mechanisms by which bisphosphonates affect osteoclasts are now evident, the exact route by which they are internalized by cells is not known. To clarify this, we synthesized a novel, fluorescently labeled analog of alendronate (AF-ALN).