Fraser P. Coxon

Cellular and molecular mechanisms of action of bisphosphonates

Bisphosphonates currently are the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor‐associated osteolysis and hypercalcemia, Pagets disease, and osteoporosis. These compounds have high affinity for calcium and therefore target to bone mineral, where they appear to be internalized selectively by bone‐resorbing osteoclasts and inhibit osteoclast function.

Protein Geranylgeranylation Is Required for Osteoclast Formation, Function, and Survival: Inhibition by Bisphosphonates and GGTI-298

Bisphosphonates are the important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Although their molecular mechanism of action has not been fully elucidated, recent studies have shown that the nitrogen‐containing bisphosphonates can inhibit protein prenylation in macrophages in vitro. In this study, we show that the nitrogen‐containing bisphosphonates risedronate, zoledronate, ibandronate, alendronate, and pamidronate (but not the non nitrogen‐containing bisphosphonates clodronate, etidronate, and tiludronate) prevent the incorporation of [14C]mevalonate into prenylated (farnesylated and geranylgeranylated) proteins in purified rabbit osteoclasts. The inhibitory effect of nitrogen‐containing bisphosphonates on bone resorption is likely to result largely from the loss of geranylgeranylated proteins rather than loss of farnesylated proteins in osteoclasts, because concentrations of GGTI‐298 (a specific inhibitor of geranylgeranyl transferase I) that inhibited protein geranylgeranylation in purified rabbit osteoclasts prevented osteoclast formation in murine bone marrow cultures, disrupted the osteoclast cytoskeleton, inhibited bone resorption, and induced apoptosis in isolated chick and rabbit osteoclasts in vitro. By contrast, concentrations of FTI‐277 (a specific inhibitor of farnesyl transferase) that prevented protein farnesylation in purified rabbit osteoclasts had little effect on osteoclast morphology or apoptosis and did not inhibit bone resorption. These results therefore show the molecular mechanism of action of nitrogen‐containing bisphosphonate drugs in osteoclasts and highlight the fundamental importance of geranylgeranylated proteins in osteoclast formation and function.

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. The considerably more potent nitrogen-containing bisphosphonates are not metabolised but potently inhibit farnesyl pyrophosphate (FPP) synthase, a key enzyme of the mevalonate pathway. This prevents the synthesis of isoprenoid lipids necessary for the post-translational prenylation of small GTPases, thereby disrupting the subcellular localisation and normal function of these essential signalling proteins. Inhibition of FPP synthase also results in the accumulation of the upstream metabolite isopentenyl diphosphate, which is incorporated into the toxic nucleotide metabolite ApppI. Together, these properties explain the ability of bisphosphonate drugs to inhibit bone resorption by disrupting osteoclast function and survival. These discoveries are also giving insights into some of the adverse effects of bisphosphonates, such as the acute phase reaction that is triggered by inhibition of FPP synthase in peripheral blood monocytes.

Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL

Autosomal recessive osteopetrosis is usually associated with normal or elevated numbers of nonfunctional osteoclasts. Here we report mutations in the gene encoding RANKL (receptor activator of nuclear factor–KB ligand) in six individuals with autosomal recessive osteopetrosis whose bone biopsy specimens lacked osteoclasts. These individuals did not show any obvious defects in immunological parameters and could not be cured by hematopoietic stem cell transplantation; however, exogenous RANKL induced formation of functional osteoclasts from their monocytes, suggesting that they could, theoretically, benefit from exogenous RANKL administration.

Bone remodelling at a glance

The bone remodelling cycle (see Poster panel “The bone remodelling cycle”) maintains the integrity of the skeleton through the balanced activities of its constituent cell types. These are the bone-forming osteoblast, a cell that produces the organic bone matrix and aids its mineralisation ([

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. From a large series of ARO patients we selected a Turkish consanguineous family with two siblings affected by ARO and hypogammaglobulinemia with no defects in known osteopetrosis genes. Sequencing of genes involved in the RANKL downstream pathway identified a homozygous mutation in the TNFRSF11A gene in both siblings. Their monocytes failed to differentiate in vitro into osteoclasts upon exposure to M-CSF and RANKL, in keeping with an osteoclast-intrinsic defect. Immunological analysis showed that their hypogammaglobulinemia was associated with impairment in immunoglobulin-secreting B cells. Investigation of other patients revealed a defect in both TNFRSF11A alleles in six additional, unrelated families. Our results indicate that TNFRSF11A mutations can cause a clinical condition in which severe ARO is associated with an immunoglobulin-production defect.

Osteopetrosis: genetics, treatment and new insights into osteoclast function.

Osteopetrosis is a genetic condition of increased bone mass, which is caused by defects in osteoclast formation and function. Both autosomal recessive and autosomal dominant forms exist, but this Review focuses on autosomal recessive osteopetrosis (ARO), also known as malignant infantile osteopetrosis. The genetic basis of this disease is now largely uncovered: mutations in TCIRG1, CLCN7, OSTM1, SNX10 and PLEKHM1 lead to osteoclast-rich ARO (in which osteoclasts are abundant but have severely impaired resorptive function), whereas mutations in TNFSF11 and TNFRSF11A lead to osteoclast-poor ARO. In osteoclast-rich ARO, impaired endosomal and lysosomal vesicle trafficking results in defective osteoclast ruffled-border formation and, hence, the inability to resorb bone and mineralized cartilage. ARO presents soon after birth and can be fatal if left untreated. However, the disease is heterogeneous in clinical presentation and often misdiagnosed. This article describes the genetics of ARO and discusses the diagnostic role of next-generation sequencing methods. The management of affected patients, including guidelines for the indication of haematopoietic stem cell transplantation (which can provide a cure for many types of ARO), are outlined. Finally, novel treatments, including preclinical data on in utero stem cell treatment, RANKL replacement therapy and denosumab therapy for hypercalcaemia are also discussed.

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). AF-ALN was rapidly internalized into intracellular vesicles in J774 macrophages and rabbit osteoclasts; uptake of AF-ALN or [14C]zoledronate was stimulated by the presence of Ca2+ and Sr2+ and could be inhibited by addition of EGTA or clodronate, both of which chelate calcium ions. Both EGTA and clodronate also prevented the bisphosphonate-induced inhibition of Rap1A prenylation, an effect that was reversed by addition of Ca2+. In J774 cells and osteoclasts, vesicular AF-ALN colocalized with dextran (but not wheat germ agglutinin or transferrin), and uptake of AF-ALN or [14C]zoledronate was inhibited by dansylcadaverine, indicating that fluid-phase endocytosis is involved in the initial internalization of bisphosphonate into vesicles. Endosomal acidification then seems to be absolutely required for exit of bisphosphonate from vesicles and entry into the cytosol, because monensin and bafilomycin A1, both inhibitors of endosomal acidification, did not inhibit vesicular uptake of AF-ALN or internalization of [14C]zoledronate but prevented the inhibitory effect of alendronate or zoledronate on Rap1A prenylation. Taken together, these results demonstrate that cellular uptake of bisphosphonate drugs requires fluid-phase endocytosis and is enhanced by Ca2+ ions, whereas transfer from endocytic vesicles into the cytosol requires endosomal acidification.

PLEKHM1 Regulates Autophagosome-Lysosome Fusion through HOPS Complex and LC3/GABARAP Proteins

The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways.

Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells

Bisphosphonates (BPs) target bone due to their high affinity for calcium ions. During osteoclastic resorption, these drugs are released from the acidified bone surface and taken up by osteoclasts, where they act by inhibiting the prenylation of small GTPases essential for osteoclast function. However, it remains unclear exactly how osteoclasts internalise BPs from bone and whether other cells in the bone microenvironment can also take up BPs from the bone surface. We have investigated this using a novel fluorescently-labelled alendronate analogue (FL-ALN), and by examining changes in protein prenylation following treatment of cells with risedronate (RIS). Confocal microscopic analysis showed that FL-ALN was efficiently internalised from solution or from the surface of dentine by resorbing osteoclasts into intracellular vesicles. Accordingly, unprenylated Rap1A accumulated to the same extent whether osteoclasts were cultured on RIS-coated dentine or with RIS in solution. By contrast, J774 macrophages internalised FL-ALN and RIS from solution, but took up comparatively little from dentine, due to their inability to resorb the mineral. Calvarial osteoblasts and MCF-7 tumour cells internalised even less FL-ALN and RIS, both from solution and from the surface of dentine. Accordingly, the viability of J774 and MCF-7 cells was drastically reduced when cultured with RIS in solution, but not when cultured on dentine pre-coated with RIS. However, when J774 macrophages were co-cultured with rabbit osteoclasts, J774 cells that were adjacent to resorbing osteoclasts frequently internalised more FL-ALN than J774 cells more distant from osteoclasts. This was possibly a result of increased availability of BP to these J774 cells due to transcytosis through osteoclasts, since FL-ALN partially co-localised with trancytosed, resorbed matrix protein within osteoclasts. In addition, J774 cells occupying resorption pits internalised more FL-ALN than those on unresorbed surfaces. These data demonstrate that osteoclasts are able to take up large amounts of BP, due to their ability to release the BP from the dentine surface during resorption. By contrast, non-resorbing cells take up only small amounts of BP that becomes available due to natural desorption from the dentine surface. However, BP uptake by non-resorbing cells can be increased when cultured in the presence of resorbing osteoclasts.