V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.     

Characterization of vacuolar-ATPase and selective inhibition of vacuolar-H(+)-ATPase in osteoclasts

V-ATPase plays important roles in controlling the extra- and intra-cellular pH in eukaryotic cell, which is most crucial for cellular processes. V-ATPases are composed of a peripheral V(1) domain responsible for ATP hydrolysis and integral V(0) domain responsible for proton translocation. Osteoclasts are multinucleated cells responsible for bone resorption and relate to many common lytic bone disorders such as osteoporosis, bone aseptic loosening, and tumor-induced bone loss. This review summarizes the structure and function of V-ATPase and its subunit, the role of V-ATPase subunits in osteoclast function, V-ATPase inhibitors for osteoclast function, and highlights the importance of V-ATPase as a potential prime target for anti-resorptive agents.     

V-ATPases的功能及其抑制剂研究进展

V-ATPases作为一类酶,在真核细胞中广泛存在。V-ATPases是一个由多个亚基组成的复合物,主要有两个结构域,分别是位于外周的V1结构域和跨膜的V0结构域。V1结构域可以通过水解ATP供能;而V0结构域是质子的通道。它们发挥作用主要是通过水解ATP供能,泵运H＋进入囊泡腔中或泵H＋出细胞外。V-ATPases定位于细胞器膜及某些特殊细胞的细胞质膜,参与骨吸收、肿瘤的侵袭及耐药等生理及病理过程,因而V-ATPases是治疗骨质疏松、糖尿病及肿瘤等人类疾病的候选分子靶标。目前有许多研究致力于发现新的潜在的特异的V-ATPase抑制剂。     

Galangin suppresses RANKL-induced osteoclastogenesis via inhibiting MAPK and NF-κB signalling pathways

Osteoclasts play a critical role in osteoporosis; thus, inhibiting osteoclastogenesis is a therapeutic strategy for osteoporosis. Galangin, a natural bioflavonoid extracted from a traditional Chinese herb, possesses a variety of biological activities, including anti-inflammation and anti-oxidation. However, its effects on osteoporosis have not been elucidated. In this study, we found that galangin treatment dose-dependently decreased osteoclastogenesis in bone marrow–derived macrophages (BMMs). Moreover, during osteoclastogenesis, osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), ATPase, H + transporting, lysosomal V0 subunit D2 (V-ATPase d2) and dendritic cell–specific transmembrane protein (DC-STAMP), were down-regulated by galangin treatment. Furthermore, the results of the pit formation assay and F-actin ring staining revealed impaired osteoclastic bone resorption in the galangin-treated group compared with that in the control group. Additionally, galangin treatment also inhibited the phosphorylation of p38 and ERK of MAPK signalling pathway, as well as downstream factors of NFATc1, C-Jun and C-Fos. Consistent with our in vitro results, galangin suppressed lipopolysaccharide (LPS)-induced bone resorption via inhibition of osteoclastogenesis. Taken together, our findings provide evidence that galangin is a promising natural compound for the treatment of osteoporosis and may be associated with the inhibition of MAPK and NF-κB signalling pathways.     

Immunohistochemical identification of a vacuolar proton pump (V-ATPase) in bone-resorbing cells of an advanced teleost species, Oreochromis niloticus

Poly-and monoclonal antibodies, raised against mammalian membrane-bound proton pump (V-ATPase) were applied to the bone-resorbing cells of Oreochromis niloticus to clarify if osteoclasts of an advanced teleost species display V-ATPase, a key enzyme in the process of bone resorption. All antibodies labelled cells at known sites of bone resorption, the endosteal bone surfaces surrounding the tooth anlagen. The best results were achieved with a monoclonal antibody (E11). Although the majority of labelled cells were flat and mononucleated, the occurrence of V-ATPase in these cells indicates that they function as active bone-resorbing cells. The monoclonal antibody E11 was also applied successfully to monocytes, cells that are believed to be related most closely to osteoclasts. The assignment of V-ATPase to boneresorbing cells of O. niloticus was confirmed by application of the additional osteoclast markers, tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant ATPase (TraATPase). Co-expression of V-ATPase, TRAP and TraATPase in fish osteoclasts is demonstrated for the first time.     

Structure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis

Excessive activity of osteoclasts becomes manifest in many common lytic bone disorders such as osteoporosis, Paget's disease, bone aseptic loosening and tumor-induced bone destruction. Vacuolar proton pump H+-adenosine triphosphatases (V-ATPases), located on the bone-apposed plasma membrane of the osteoclast, are imperative for the function of osteoclasts, and thus are a potential molecular target for the development of novel anti-resorptive agents. To date, the V-ATPases core structure has been well modeled and consists of two distinct functional domains, the V1 (A, B1, B2, C1, C2, D, E1, E2, F, G1, G2, G3, and H subunits) and V0 (a1, a2, a3, a4, d1, d2, c, c' e1, e2 subunits) as well as the accessory subunits ac45 and M8-9. However, the exact configuration of osteoclast specific V-ATPases remains to be established. Inactivation of subunit a3 leads to osteopetrosis in both mice and man because of non-functional osteoclasts that are capable of acidifying the extracellular resorption lacuna. On the other hand, inactivation of subunits c, d1 and ac45 results in early embryonic lethality, indicating that certain subunits, such as a3, are more specific to osteoclast function than others. In osteoclasts, V-ATPases also cooperate with chloride channel protein CLC-7 to acidify the resorption lacuna. In addition, development of V-ATPases inhibitors such as bafilomycin A1, SB 242784 and FR167356 that selectively target osteoclast specific V-ATPases remains a challenge. Understanding the molecular and cellular mechanisms by which specific subunits of V-ATPase regulate osteoclast function might facilitate the development of novel and selective inhibitors for the treatment of lytic bone disorders. This review summarizes recent research developments in V-ATPases with particular emphasis on osteoclast biology.     

Age-associated Iron Accumulation in Bone: Implications for Postmenopausal Osteoporosis and a New Target for Prevention and Treatment by Chelation

Iron accumulation in tissues is believed to be a characteristic of aged humans and a risk factor for some chronic diseases. However, it is not known whether age-associated iron accumulation is part of the pathogenesis of postmenopausal osteoporosis that affects approximately one out three women worldwide. Here, we confirmed that this accumulation of iron was associated with osteopenia in ovariectomized (OVX) rats (a model of peri- and postmenopausal osteoporosis due to estrogen deficiency). To further investigate whether the increased iron level plays a causal role in the onset of bone loss, we treated OVX rats with an orally active and bone targeted chelator that prevented iron accumulation in their skeletal tissues. The results showed that this treatment mitigated the loss of bone mass and the deterioration of bone micro-architecture. We also found that one possible mechanism of the protective action of iron chelation was to significantly reduce bone resorption. Thus, these findings provide a novel target and a potentially useful therapeutic strategy for the prevention and treatment of postmenopausal osteoporosis and perhaps other age-related diseases.     

Osteoporosis: the current status of mesenchymal stem cell-based therapy

Osteoporosis, or bone loss, is a progressive, systemic skeletal disease that affects millions of people worldwide. Osteoporosis is generally age related, and it is underdiagnosed because it remains asymptomatic for several years until the development of fractures that confine daily life activities, particularly in elderly people. Most patients with osteoporotic fractures become bedridden and are in a life-threatening state. The consequences of fracture can be devastating, leading to substantial morbidity and mortality of the patients. The normal physiologic process of bone remodeling involves a balance between bone resorption and bone formation during early adulthood. In osteoporosis, this process becomes imbalanced, resulting in gradual losses of bone mass and density due to enhanced bone resorption and/or inadequate bone formation. Several growth factors underlying age-related osteoporosis and their signaling pathways have been identified, such as osteoprotegerin (OPG)/receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL), bone morphogenetic protein (BMP), wingless-type MMTV integration site family (Wnt) proteins and signaling through parathyroid hormone receptors. In addition, the pathogenesis of osteoporosis has been connected to genetics. The current treatment of osteoporosis predominantly consists of antiresorptive and anabolic agents; however, the serious adverse effects of using these drugs are of concern. Cell-based replacement therapy via the use of mesenchymal stem cells (MSCs) may become one of the strategies for osteoporosis treatment in the future.     

Prospect of mesenchymal stem cells in therapy of osteoporosis: A review

Osteoporosis is a systemic skeletal disease associated with reduced bone strong point that results in raised fracture risk, with decreased bone strength, leading to reduced bone mineral density and poor bone quality. It is the most common in older females but some men are also at high risk. Although considered as a predictable result of aging, it is can be avoidable and treatable. The existing treatment of osteoporosis mainly contains antiresorptive and anabolic agents. In spite of these improvements, concerns around unusual side-effects of antiresorptive drugs, and the lack of perfect confirmation in maintenance of their long-standing effectiveness is bring about many patients not receiving these drugs. Over the years, the stem cell-based therapy has attained substantial clinical consideration because of its potential to treat numerous diseases. The stem cell therapy has been recommended as a probable therapeutic approach for patients with osteoporosis. Even though the concept of stem cell-based therapy for osteoporosis has caught substantial attention, no clinical trial has been published on humans. The cell studies based on osteoporosis are primarily focused on osteoclastic activity and bone resorption procedures. Earlier, it was on osteoblastogenesis and in recent times, on the differentiation probable of mesenchymal stem cells. In this review, we have summarized the therapeutic role of stem cell-based strategy in osteoporosis.     

PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism

The vacuolar ATPase (V-ATPase) is a 1MDa transmembrane proton pump that operates via a rotary mechanism fuelled by ATP. Essential for eukaryotic cell homeostasis, it plays central roles in bone remodeling and tumor invasiveness, making it a key therapeutic target. Its importance in arthropod physiology also makes it a promising pesticide target. The major challenge in designing lead compounds against the V-ATPase is its ubiquitous nature, such that any therapeutic must be capable of targeting particular isoforms. Here, we have characterized the binding site on the V-ATPase of pea albumin 1b (PA1b), a small cystine knot protein that shows exquisitely selective inhibition of insect V-ATPases. Electron microscopy shows that PA1b binding occurs across a range of equivalent sites on the c ring of the membrane domain. In the presence of Mg·ATP, PA1b localizes to a single site, distant from subunit a, which is predicted to be the interface for other inhibitors. Photoaffinity labeling studies show radiolabeling of subunits c and e. In addition, weevil resistance to PA1b is correlated with bafilomycin resistance, caused by mutation of subunit c. The data indicate a binding site to which both subunits c and e contribute and inhibition that involves locking the c ring rotor to a static subunit e and not subunit a. This has implications for understanding the V-ATPase mechanism and that of inhibitors with therapeutic or pesticidal potential. It also provides the first evidence for the position of subunit e within the complex.     

The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function

Vacuolar-type H+-ATPase (V-ATPase)-driven proton pumping and organellar acidification is essential for vesicular trafficking along both the exocytotic and endocytotic pathways of eukaryotic cells. Deficient function of V-ATPase and defects of vesicular acidification have been recently recognized as important mechanisms in a variety of human diseases and are emerging as potential therapeutic targets. In the past few years, significant progress has been made in our understanding of function, regulation, and the cell biological role of V-ATPase. Here, we will review these studies with emphasis on novel direct roles of V-ATPase in the regulation of vesicular trafficking events.     

Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts

Bone resorption relies on the extracellular acidification function of V-ATPase (vacuolar-type proton-translocating ATPase) proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of the osteoclast-specific ruffled border V-ATPases remains largely unknown. In the present study, we found that the V-ATPase subunit Atp6v1c1 (C1) is highly expressed in osteoclasts, whereas subunits Atp6v1c2a (C2a) and Atp6v1c2b (C2b) are not. The expression level of C1 is highly induced by RANKL [receptor activator for NF-kappaB (nuclear factor kappaB) ligand] during osteoclast differentiation; C1 interacts with Atp6v0a3 (a3) and is mainly localized on the ruffled border of activated osteoclasts. The results of the present study show for the first time that C1-silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption, whereas cell differentiation did not appear to be affected, which is similar to a3 silencing. The F-actin (filamentous actin) ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3(-/-) osteoclasts. C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. In addition, C1 co-localized with F-actin in the cytoplasm; however, the co-localization chiefly shifted to the cell periphery of mature osteoclasts. The present study demonstrates that Atp6v1c1 is an essential component of the osteoclast proton pump at the osteoclast ruffled border and that it may regulate F-actin ring formation in osteoclast activation.     

The vacuolar (H+)-ATPase: subunit arrangement and in vivo regulation

The V-ATPases are responsible for acidification of intracellular compartments and proton transport across the plasma membrane. They play an important role in both normal processes, such as membrane traffic, protein degradation, urinary acidification, and bone resorption, as well as various disease processes, such as viral infection, toxin killing, osteoporosis, and tumor metastasis. V-ATPases contain a peripheral domain (V₁) that carries out ATP hydrolysis and an integral domain (V₀) responsible for proton transport. V-ATPases operate by a rotary mechanism involving both a central rotary stalk and a peripheral stalk that serves as a stator. Cysteine-mediated cross-linking has been used to localize subunits within the V-ATPase complex and to investigate the helical interactions between subunits within the integral V₀ domain. An essential property of the V-ATPases is the ability to regulate their activity in vivo. An important mechanism of regulating V-ATPase activity is reversible dissociation of the complex into its component V₁ and V₀ domains. The dependence of reversible dissociation on subunit isoforms and cellular environment has been investigated. Qi and Wang contributed equally to this work.     

Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology

The acidity of intracellular compartments and the extracellular environment is crucial to various cellular processes, including membrane trafficking, protein degradation, bone resorption and sperm maturation. At the heart of regulating acidity are the vacuolar (V-)ATPases--large, multisubunit complexes that function as ATP-driven proton pumps. Their activity is controlled by regulating the assembly of the V-ATPase complex or by the dynamic regulation of V-ATPase expression on membrane surfaces. The V-ATPases have been implicated in a number of diseases and, coupled with their complex isoform composition, represent attractive and potentially highly specific drug targets.     

Anti-osteopontin monoclonal antibody prevents ovariectomy-induced osteoporosis in mice by promotion of osteoclast apoptosis

Osteopontin (OPN) is abundant in mineralized tissues and has long been implicated in bone remodeling. However, the therapeutic effect of targeting OPN in bone loss diseases and the underlying molecular mechanism remain largely unknown. Here, we reported that anti-OPN mAb (23C3) could protect against ovariectomy-induced osteoporosis in mice, demonstrated by microcomputed tomography analysis and histopathology evaluation. In vitro assay showed that 23C3 mAb reduced osteoclasts (OCs)-mediated bone resorption through promotion of mature OC apoptosis. Thus, the study has important implications for understanding the role of OPN in OC bone resorption and survival, and OPN antagonists may have therapeutic potential for osteoporosis and other osteopenic diseases.     

New Treatment Modalities in Osteoporosis

ObjectiveTo describe recently discovered agents for the management of osteoporosis.MethodsA literature review (PubMed search) was conducted to identify agents at various stages of development for osteoporosis treatment. Agents under study or review for approval were included.ResultsIn menopause, bone remodeling is increased, and agents that suppress bone resorption can stabilize bone mass. In contrast, agents that target the osteoblast can increase bone formation and bone mass. Novel antiresorptive agents can target the formation or the activity of osteoclasts. They include denosumab, an antibody to receptor activated nuclear factor kB; new selective estrogen receptor modulators, such as bazedoxifene; and cathepsin K inhibitors, such as odanacatib. Src kinase inhibitors are in the early phases of development. Parathyroid hormone is the only approved anabolic agent for the treatment of osteoporosis. Novel anabolic therapies for osteoporosis may include the use of factors with anabolic properties for bone or the neutralization of growth factor antagonists. Recent discoveries have demonstrated that the Wnt/β-catenin signaling pathway has a central role in osteoblastic cell differentiation. Antibodies to Wnt antagonists, such as sclerostin, are under development as new therapeutic approaches for osteoporosis. Anabolic therapies have the potential to enhance bone mass, but their long-term safety must be proven.ConclusionsNew developments in the treatment of osteoporosis include novel antiresorptive and anabolic agents. Their success will depend on their long-term effectiveness and safety profile. (Endocr Pract. 2010;16:855-863)     

Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.     

Function, structure and regulation of the vacuolar (H+)-ATPases

The vacuolar ATPases (or V-ATPases) are ATP-driven proton pumps that function to both acidify intracellular compartments and to transport protons across the plasma membrane. Intracellular V-ATPases function in such normal cellular processes as receptor-mediated endocytosis, intracellular membrane traffic, prohormone processing, protein degradation and neurotransmitter uptake, as well as in disease processes, including infection by influenza and other viruses and killing of cells by anthrax and diphtheria toxin. Plasma membrane V-ATPases are important in such physiological processes as urinary acidification, bone resorption and sperm maturation as well as in human diseases, including osteopetrosis, renal tubular acidosis and tumor metastasis. V-ATPases are large multi-subunit complexes composed of a peripheral domain (V₁) responsible for hydrolysis of ATP and an integral domain (V₀) that carries out proton transport. Proton transport is coupled to ATP hydrolysis by a rotary mechanism. V-ATPase activity is regulated in vivo using a number of mechanisms, including reversible dissociation of the V₁ and V₀ domains, changes in coupling efficiency of proton transport and ATP hydrolysis and changes in pump density through reversible fusion of V-ATPase containing vesicles. V-ATPases are emerging as potential drug targets in treating a number of human diseases including osteoporosis and cancer.     

Inhibition of receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation and bone resorption by Gryllus bimaculatus extract: An in vitro study

Excessive bone-resorbing osteoclast activity during bone remodeling is a major feature of bone diseases, such as osteoporosis. Therefore, the inhibition of osteoclast formation and bone resorption can be an effective therapeutic target for various bone diseases. Gryllus biomaculatus (GB) has recently been approved as an alternative food source because of its high nutritional value and environmental sustainability. Traditionally, GB has been known to have various pharmacological properties, including antipyretic and blood pressure-lowering activity, and it has recently been reported to have various biological activities, including protective effects against inflammation, oxidative stress, insulin resistance, and alcohol-induced liver injury. However, the effect of GB on osteoclast differentiation and bone metabolism has not yet been demonstrated. In this study, we confirmed the inhibitory effect of GB extract (GBE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. To determine the effect of GBE on RANKL-induced osteoclast differentiation and function, we performed TRAP and F-actin staining, as well as a bone-resorbing assay. The intracellular mechanisms of GBE responsible for the regulation of osteoclastogenesis were revealed by Western blot analysis and quantitative real-time polymerase chain reaction. We investigated the relationship between GBE and expression of osteoclast-specific molecules to further elucidate the underlying mechanisms. It was found that GBE significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt, p38, JNK, and ERK, as well as Btk-PLCγ2 signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos, NFATc1, and osteoclastogenesis-specific marker genes. Additionally, GBE inhibited the formation of F-actin ring-positive osteoclasts and bone resorption activity of mature osteoclasts. Our findings suggest that GBE is a potential functional food and therapeutic candidate for bone diseases involving osteoclasts.     

Proteomic identification of the TRAF6 regulation of vacuolar ATPase for osteoclast function

Osteoclasts are cells specialized for bone resorption. For osteoclast activation, tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role. To find new molecules that bind TRAF6 and have a function in osteoclast activation, we employed a proteomic approach. TRAF6-binding proteins were purified from osteoclast cell lysates by affinity chromatography and their identity was disclosed by MS. The identified proteins included several heat shock proteins, actin and actin-binding proteins, and vacuolar ATPase (V-ATPase). V-ATPase, documented for a great increase in expression during osteoclast differentiation, is an important enzyme for osteoclast function; it transports proton to resorption lacunae for hydroxyapatite dissolution. The binding of V-ATPase with TRAF6 was confirmed both in vitro by GST pull-down assays and in osteoclasts by co-immunoprecipitation and confocal microscopy experiments. In addition, the V-ATPase activity associated with TRAF6 increased in osteoclasts stimulated with receptor activator of nuclear factor kappaB ligand (RANKL). Furthermore, a dominant-negative form of TRAF6 abrogated the RANKL stimulation of V-ATPase activity. Our study identified V-ATPase as a TRAF6-binding protein using a proteomics strategy and proved a direct link between these two important molecules for osteoclast function.