Atorvastatin-mediated inhibition of prenylation of Rab27b and Rap1a in platelets attenuates their prothrombotic capacity and modulates clot structure.

Statins inhibit the mevalonate pathway by impairing protein prenylation via depletion of lipid geranylgeranyl diphosphate (GGPP). Rab27b and Rap1a are small GTPase proteins involved in dense granule secretion, platelet activation, and regulation. We analyzed the impact of statins on prenylation of Rab27b and Rap1a in platelets and the downstream effects on fibrin clot properties. Whole blood thromboelastography revealed that atorvastatin (ATV) delayed clot formation time (P < .005) and attenuated clot firmness (P < .005). ATV pre-treatment inhibited platelet aggregation and clot retraction. Binding of fibrinogen and P-selectin exposure on stimulated platelets was significantly lower following pre-treatment with ATV (P < .05). Confocal microscopy revealed that ATV significantly altered the structure of platelet-rich plasma clots, consistent with the reduced fibrinogen binding. ATV enhanced lysis of Chandler model thrombi 1.4-fold versus control (P < .05). Western blotting revealed that ATV induced a dose-dependent accumulation of unprenylated Rab27b and Rap1a in the platelet membrane. ATV dose-dependently inhibited ADP release from activated platelets. Exogenous GGPP rescued the prenylation of Rab27b and Rap1a, and partially restored the ADP release defect, suggesting these changes arise from reduced prenylation of Rab27b. These data demonstrate that statins attenuate platelet aggregation, degranulation, and binding of fibrinogen thereby having a significant impact on clot contraction and structure.

What is the context? Statins such as Atorvastatin (ATV) are 3-hydroxy, 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the cholesterol biosynthetic pathway to lower total serum levels and LDL-cholesterol.The cholesterol pathway also provides a supply of isoprenoids (farnesyl and geranylgeranyl) for the prenylation of signaling molecules, which include the families of Ras and Rho small GTPases.Prenyl groups provide a membrane anchor that is essential for the correct membrane localization and function of these proteins.Statins deplete cells of lipid geranylgeranyl diphosphate (GGPP) thereby inhibiting progression of the mevalonate pathway and prenylation of proteins.Rab27b and Rap1 are small GTPase proteins in platelets that are involved in the secretion of platelet granules and integrin activation.What is new?In this study, we found that ATV impairs prenylation of Rab27b and Rap1a and attenuates platelet function.These effects were partially rescued by GGPP, indicating the involvement of the mevalonate pathway.Platelet aggregation and degranulation was significantly attenuated by ATV.The impact of statins on platelet function altered clot formation, structure and contraction generating a clot that was more susceptible to degradation.What is the impact?This study demonstrates a novel mechanism whereby statins alter platelet responses and ultimately clot structure and stability.

SNX10 gene mutation leading to osteopetrosis with dysfunctional osteoclasts.

Autosomal recessive osteopetrosis (ARO) is a heterogeneous disorder, characterized by defective osteoclastic resorption of bone that results in increased bone density. We have studied nine individuals with an intermediate form of ARO, from the county of Västerbotten in Northern Sweden. All afflicted individuals had an onset in early infancy with optic atrophy, and in four patients anemia was present at diagnosis. Tonsillar herniation, foramen magnum stenosis, and severe osteomyelitis of the jaw were common clinical features. Whole exome sequencing, verified by Sanger sequencing, identified a splice site mutation c.212 + 1 G > T in the SNX10 gene encoding sorting nexin 10. Sequence analysis of the SNX10 transcript in patients revealed activation of a cryptic splice site in intron 4 resulting in a frame shift and a premature stop (p.S66Nfs * 15). Haplotype analysis showed that all cases originated from a single mutational event, and the age of the mutation was estimated to be approximately 950 years. Functional analysis of osteoclast progenitors isolated from peripheral blood of patients revealed that stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) resulted in a robust formation of large, multinucleated osteoclasts which generated sealing zones; however these osteoclasts exhibited defective ruffled borders and were unable to resorb bone in vitro.

Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile Imaging Toolkit for Applications in Bone Biology and Biomedicine.

A bone imaging toolkit of 21 fluorescent probes with variable spectroscopic properties, bone mineral binding affinities, and antiprenylation activities has been created, including a novel linking strategy. The linking chemistry allows attachment of a diverse selection of dyes fluorescent in the visible to near-infrared range to any of the three clinically important heterocyclic bisphosphonate bone drugs (risedronate, zoledronate, and minodronate or their analogues). The resultant suite of conjugates offers multiple options to "mix and match" parent drug structure, fluorescence emission wavelength, relative bone affinity, and presence or absence of antiprenylation activity, for bone-related imaging applications.

PLEKHM1 regulates Salmonella-containing vacuole biogenesis and infection.

The host endolysosomal compartment is often manipulated by intracellular bacterial pathogens. Salmonella (Salmonella enterica serovar Typhimurium) secrete numerous effector proteins, including SifA, through a specialized type III secretion system to hijack the host endosomal system and generate the Salmonella-containing vacuole (SCV). To form this replicative niche, Salmonella targets the Rab7 GTPase to recruit host membranes through largely unknown mechanisms. We show that Pleckstrin homology domain-containing protein family member 1 (PLEKHM1), a lysosomal adaptor, is targeted by Salmonella through direct interaction with SifA. By binding the PLEKHM1 PH2 domain, Salmonella utilize a complex containing PLEKHM1, Rab7, and the HOPS tethering complex to mobilize phagolysosomal membranes to the SCV. Depletion of PLEKHM1 causes a profound defect in SCV morphology with multiple bacteria accumulating in enlarged structures and significantly dampens Salmonella proliferation in multiple cell types and mice. Thus, PLEKHM1 provides a critical interface between pathogenic infection and the host endolysosomal system.

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.

Synthesis and characterization of novel phosphonocarboxylate inhibitors of RGGT.

Phosphonocarboxylate (PC) analogs of the anti-osteoporotic drugs, bisphosphonates, represent the first class of selective inhibitors of Rab geranylgeranyl transferase (RabGGTase, RGGT), an enzyme implicated in several diseases including ovarian, breast and skin cancer. Here we present the synthesis and biological characterization of an extended set of this class of compounds, including lipophilic derivatives of the known RGGT inhibitors. From this new panel of PCs, we have identified an inhibitor of RGGT that is of similar potency as the most active published phosphonocarboxylate, but of higher selectivity towards this enzyme compared to prenyl pyrophosphate synthases. New insights into structural requirements are also presented, showing that only PC analogs of the most potent 3rd generation bisphosphonates inhibit RGGT. In addition, the first phosphonocarboxylate-derived GGPPS inhibitor is reported.

Role of vesicular trafficking in skeletal dynamics.

Vesicular trafficking is critical for the function of bone cells, exemplified by bone diseases such as osteopetrosis, which frequently results from defects in this process. Recent work has further dissected the role of the endolysosomal system in both bone formation by osteoblasts and bone resorption by osteoclasts. This pathway also plays an important role in the communication between these and other cells in bone, through trafficking and degradation of growth factors and their receptors, and microvesicle release. In addition, a crucial role for autophagy in bone remodelling and bone disease is beginning to emerge. These insights into the molecular control of bone remodelling raise the possibility of developing novel therapeutics for bone diseases designed to target specific aspects of this process.

Jaw bone marrow-derived osteoclast precursors internalize more bisphosphonate than long-bone marrow precursors.

Bisphosphonates (BPs) are widely used in the treatment of several bone diseases, such as osteoporosis and cancers that have metastasized to bone, by virtue of their ability to inhibit osteoclastic bone resorption. Previously, it was shown that osteoclasts present at different bone sites have different characteristics. We hypothesized that BPs could have distinct effects on different populations of osteoclasts and their precursors, for example as a result of a different capacity to endocytose the drugs. To investigate this, bone marrow cells were isolated from jaw and long bone from mice and the cells were primed to differentiate into osteoclasts with the cytokines M-CSF and RANKL. Before fusion occurred, cells were incubated with fluorescein-risedronate (FAM-RIS) for 4 or 24h and uptake was determined by flow cytometry. We found that cultures obtained from the jaw internalized 1.7 to 2.5 times more FAM-RIS than long-bone cultures, both after 4 and 24h, and accordingly jaw osteoclasts were more susceptible to inhibition of prenylation of Rap1a after treatment with BPs for 24h. Surprisingly, differences in BP uptake did not differentially affect osteoclastogenesis. This suggests that jaw osteoclast precursors are less sensitive to bisphosphonates after internalization. This was supported by the finding that gene expression of the anti-apoptotic genes Bcl-2 and Bcl-xL was higher in jaw cells than long bone cells, suggesting that the jaw cells might be more resistant to BP-induced apoptosis. Our findings suggest that bisphosphonates have distinct effects on both populations of osteoclast precursors and support previous findings that osteoclasts and precursors are bone-site specific. This study may help to provide more insights into bone-site-specific responses to bisphosphonates.

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.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton.

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)ß(3)) at the desired time following in vivo administration of the drug. Since osteoclasts are abundant in newborn rabbits, sufficient numbers of osteoclasts can be retrieved using this method for molecular and biochemical analyses.

Generation of human osteoclasts from peripheral blood.

Osteoclasts are multi-nucleated cells that have the unique ability to resorb calcified bone matrix. They derive from haematopoietic precursor cells, and can be generated in vitro by stimulation of peripheral blood mononuclear cells with the cytokines M-CSF and RANKL. In this chapter, we describe the method for generating human osteoclast from peripheral blood or buffy coats, as well as methods for studying both the differentiation and resorbing activity of these cells.

Fluorescence imaging of osteoclasts using confocal microscopy.

In order to understand osteoclast cell biology, it is necessary to culture these cells on a physiological -substrate that they can resorb in vitro, such as bone or dentine. However, this creates problems for analysis by fluorescence microscopy, due to the depth of the sample under investigation. By virtue of its optical sectioning capabilities, confocal microscopy is ideal for analysis of such samples, enabling precise intracellular localisation of proteins in resorbing osteoclasts to be determined. Moreover, by taking a series of images in the axial dimension, it is possible to create axial section views and to reconstruct 3D images of the osteoclasts, enabling the spatial organisation of the structures of interest to be more easily discerned.

Synthesis and characterization of novel fluorescent nitrogen-containing bisphosphonate imaging probes for bone active drugs.

Progress in the synthesis of novel fluorescent conjugates of N-heterocyclic bisphosphonate drugs and related analogues, together with some recent applications of these compounds as imaging probes, are briefly discussed.

Synthesis, stereochemistry and SAR of a series of minodronate analogues as RGGT inhibitors.

Phosphonocarboxylate (PC) analogues of bisphosphonates are of interest due to their selective inhibition of a key enzyme in the mevalonate pathway, Rab geranylgeranyl transferase (RGGT). The dextrarotatory enantiomer of 2-hydroxy-3-(imidazo[1,2-a]pyridin-3-yl)-2-phosphonopropanoic acid (3-IPEHPC, 1) is the most potent PC-type RGGT inhibitor thus far identified. The absolute configuration of (+)-1 in the active site complex has remained unknown due to difficulties in obtaining RGGT inhibitor complex crystals suitable for X-ray diffraction analysis. However, we have now succeeded in crystallizing (-)-1 and here report its absolute configuration (AC) obtained by X-ray crystallography, thus also defining the AC of (+)-1. An Autodock Vina 1.1 computer modeling study of (+)-1 in the active site of modified RGGT binding GGPP (3DSV) identifies stereochemistry-dependent interactions that could account for the potency of (+)-1 and supports the hypothesis that this type of inhibitor binds at the TAG tunnel, inhibiting the second geranylgeranylation step. We also report a convenient (31)P NMR method to determine enantiomeric excess of 1 and its pyridyl analogue 2, using α- and ß-cyclodextrins as chiral solvating agents, and describe the synthesis of a small series of 1 α-X (X = H, F, Cl, Br; 7a-d) analogues to assess the contribution of the α-OH group to activity at enzyme and cellular levels. The IC(50) of 1 was 5-10× lower than 7a-d, and the LED for inhibition of Rab11 prenylation in vitro was 2-8× lower than for 7a-d. However, in a viability reduction assay with J774 cells, 1 and 7b had similar IC(50) values, ~10× lower than those of 7a and 7c-d.

Impaired prenylation of Rab GTPases in the gunmetal mouse causes defects in bone cell function.

Vesicular trafficking is crucial for bone resorption by osteoclasts, in particular for formation of the ruffled border membrane and for removal of the resultant bone degradation products by transcytosis. These processes are regulated by Rab family GTPases, whose activity is dependent on post-translational prenylation by Rab geranylgeranyl transferase (RGGT). Specific pharmacological inhibition of RGGT inhibits bone resorption in vitro and in vivo, illustrating the importance of Rab prenylation for osteoclast function. The gunmetal (gm/gm) mouse bears a mutation in the catalytic subunit of RGGT, causing a loss of 75% of the activity of this enzyme and hence hypoprenylation of several Rabs in melanocytes, platelets and cytotoxic T cells. We have now found that prenylation of several Rab proteins is also defective in gm/gm osteoclasts. Moreover, while osteoclast formation and cytoskeletal polarization occurs normally, gm/gm osteoclasts exhibit a substantial reduction in resorptive activity in vitro compared with osteoclasts from +/gm mice, which do not have a prenylation defect. Surprisingly, rather than the osteosclerosis that would be expected to result from defective osteoclast function in vivo, gm/gm mice exhibited a slightly lower bone mass than +/gm mice, indicating that defects in other cell types, such as osteoblasts, in which hypoprenylation of Rabs was also detected, may contribute to the phenotype. However, gm/gm mice were partially protected from ovariectomy-induced bone loss, suggesting that levels of Rab prenylation in gm/gm osteoclasts may be sufficient to maintain normal physiological levels of activity, but not pathological levels of bone resorption in vivo.

The regulation of osteoclast function and bone resorption by small GTPases.

Osteoclasts are multinucleated cells that are responsible for resorption of bone, and increased activity of these cells is associated with several common bone diseases, including postmenopausal osteoporosis. Upon adhesion to bone, osteoclasts become polarized and reorganise their cytoskeleton and membrane to form unique domains including the sealing zone (SZ), which is a dense ring of F-actin-rich podosomes delimiting the ruffled border (RB), where protons and proteases are secreted to demineralise and degrade the bone matrix, respectively. These processes are dependent on the activity of small GTPases. Rho GTPases are well known to control the organization of F-actin and adhesion structures of different cell types, affecting subsequently their migration. In osteoclasts, RhoA, Rac, Cdc42, RhoU and also Arf6 regulate podosome assembly and their organization into the SZ. By contrast, the formation of the RB involves vesicular trafficking pathways that are regulated by the Rab family of GTPases, in particular lysosomal Rab7. Finally, osteoclast survival is dependent on the activity of Ras GTPases. The correct function of almost all these GTPases is absolutely dependent on post-translational prenylation, which enables them to localize to specific target membranes. Bisphosphonate drugs, which are widely used in the treatment of bone diseases such as osteoporosis, act by preventing the prenylation of small GTPases, resulting in the loss of the SZ and RB and therefore inhibition of osteoclast activity, as well as inducing osteoclast apoptosis. In this review we summarize current understanding of the role of specific prenylated small GTPases in osteoclast polarization, function and survival.

The gunmetal mouse reveals Rab geranylgeranyl transferase to be the major molecular target of phosphonocarboxylate analogues of bisphosphonates.

The described ability of phosphonocarboxylate analogues of bisphosphonates (BPs) to inhibit Rab geranylgeranyl transferase (RGGT) is thought to be the mechanism underlying their cellular effects, including their ability to reduce macrophage cell viability and to inhibit osteoclast-mediated resorption. However, until now the possibility that at least some of the effects of these drugs may be mediated through other targets has not been excluded. Since RGGT is the most distal enzyme in the process of Rab prenylation, it has not proved possible to confirm the mechanism underlying the effects of these drugs by adding back downstream intermediates of the mevalonate pathway, the approach used to demonstrate that bisphosphonates act through this pathway. We now confirm that RGGT is the major pharmacological target of phosphonocarboxylates by using several alternative approaches. Firstly, analysis of several different phosphonocarboxylate drugs demonstrates a very good correlation between the ability of these drugs to inhibit RGGT with their ability to: (a) reduce macrophage cell viability; (b) induce apoptosis; and (c) induce vacuolation in rabbit osteoclasts. Secondly, we have found that cells from the gunmetal (gm/gm) mouse, which bear a homozygous mutation in RGGT that results in ~80% reduced activity of this enzyme compared to wild-type or heterozygous mice, are more sensitive to the effects of active phosphonocarboxylates (including reducing macrophage cell viability, inhibiting osteoclast formation and inhibiting fluid-phase endocytosis), confirming that these effects are mediated through inhibition of RGGT. In conclusion, these data demonstrate that all of the pharmacological effects of phosphonocarboxylates found thus far appear to be mediated through the specific inhibition of RGGT, highlighting the potential therapeutic value of this class of drugs.

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.