Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.     

Membrane trafficking in osteoblasts and osteoclasts: new avenues for understanding and treating skeletal diseases

The endocytic and exocytic/secretory pathways are two major intracellular membrane trafficking routes that regulate numerous cellular functions in a variety of cell types. Osteoblasts and osteoclasts, two major bone cells responsible for bone remodeling and homeostasis, are no exceptions. During the past few years, emerging evidence has pinpointed a critical role for endocytic and secretory pathways in osteoblast and osteoclast differentiation and function. The endosomal membrane provides a platform to integrate bone tropic signals of hormones and growth factors in osteoblasts. In osteoclasts, endocytosis, followed by transcytosis, of degraded bone matrix promotes bone resorption. Secretory pathways, especially lysosome secretion, not only participate in bone matrix deposition by osteoblasts and degradation of mineralized bone matrix by osteoclasts; they may also be involved in the coupling of bone resorption and bone formation during bone remodeling. More importantly, mutations in genes encoding regulatory factors within the endocytic and secretory pathways have been identified as causes for bone diseases. Identification of the molecular mechanisms of these genes in bone cells may provide new therapeutic targets for skeletal disorders.     

Proteomic profile of osteoclast membrane proteins: identification of Na+/H+ exchanger domain containing 2 and its role in osteoclast fusion

Osteoclast formation and bone resorption are multiple processes that involve the participation of specialized membrane structures and their associated proteins. In this study, we used an MS to analyze the profile of proteins associated with osteoclast membranes and focused on the function of channel proteins in osteoclast differentiation and function. We filtered out with a SEQUEST score greater than 10 and a peptide hit number of more than 2, resulting in the identification of 499 proteins that were commonly found in both macrophages and osteoclasts, 96 proteins selectively found in osteoclasts, and 179 proteins selectively found in macrophages. The proteins that were selectively found in osteoclasts were classified based on their localizations: plasma membrane (17%), ER/Golgi and lysosome/endosome (15%), mitochondrion (18%), nucleus (13%), cytosol (19%), and unknown (18%). Proteins associated with osteoclast function such as v-ATPase, IGF2R, TRAP, and cathepsin K were found in osteoclasts as previously shown. We found several ion channel proteins such as Ank and Nhedc2 and signaling molecules such as Dock5 and RAB-10 in osteoclasts. Inhibition of the Na(+)/H(+) exchanger family by amiloride suppressed RANKL-induced osteoclast fusion and bone resorption. In addition, shRNA for Nhedc2 inhibited osteoclast differentiation. Our results provide a proteomic profile of osteoclast membrane proteins and identify Nhedc2, which is probably associated with proton transport in osteoclasts, as a regulator of osteoclast function.     

Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism

Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.     

Synaptotagmin VII regulates bone remodeling by modulating osteoclast and osteoblast secretion

Maintenance of bone mass and integrity requires a tight balance between resorption by osteoclasts and formation by osteoblasts. Exocytosis of functional proteins is a prerequisite for the activity of both cells. In the present study, we show that synaptotagmin VII, a calcium sensor protein that regulates exocytosis, is associated with lysosomes in osteoclasts and bone matrix protein-containing vesicles in osteoblasts. Absence of synaptotagmin VII inhibits cathepsin K secretion and formation of the ruffled border in osteoclasts and bone matrix protein deposition in osteoblasts, without affecting the differentiation of either cell. Reflecting these in vitro findings, synaptotagmin VII-deficient mice are osteopenic due to impaired bone resorption and formation. Therefore, synaptotagmin VII plays an important role in bone remodeling and homeostasis by modulating secretory pathways functionally important in osteoclasts and osteoblasts.     

SNX10 is required for osteoclast formation and resorption activity

RANKL-stimulation of osteoclast precursors results in up-regulation of genes involved in the process of differentiation and activation. In this report we describe the expression and functional characterization of Sorting Nexin 10 (snx10). Snx10 belongs to the sorting nexin (SNX) family, a diverse group of proteins with a common feature: the PX domain, which is involved in membrane trafficking and cargo sorting in endosomes. Snx10 is strongly up-regulated during RANKL-induced osteoclast differentiation in vitro and expressed in osteoclasts in vivo. qPCR analysis confirmed a significant increase in the expression of snx10 in in vitro-derived osteoclasts, as well as in femur and calvaria. Immunohistochemical analysis of mouse embryo sections showed expression in long bone, calvariae, and developing teeth. The expression was limited to cells that also expressed TRAP, demonstrating osteoclastic localization. Confocal immunofluorescence and subcellular fractionation analysis revealed Snx10 localization in the nucleus and in the endoplasmic reticulum (ER). To study a possible role for snx10 in osteoclast differentiation and function we silenced snx10 expression and found that snx10 silencing inhibited RANKL-induced osteoclast formation and osteoclast resorption on hydroxyapatite. Silencing also inhibited TRAP secretion. Taken together, these results confirm that snx10 is expressed in osteoclasts and is required for osteoclast differentiation and activity in vitro. Since inhibition of vesicular trafficking is essential for osteoclast formation and activity and SNX10 is involved in intracellular vesicular trafficking, these studies may identify a new candidate gene involved in the development of human bone diseases including osteoporosis.     

Nitric oxide regulation of cGMP production in osteoclasts

Bone resorption by osteoclasts is modified by agents that affect cyclic guanosine monophosphate (cGMP), but their relative physiological roles, and what components of the process are present in osteoclasts or require accessory cells such as osteoblasts, are unclear. We studied cGMP regulation in avian osteoclasts, and in particular the roles of nitric oxide and natriuretic peptides, to clarify the mechanisms involved. C-type natriuretic peptide drives a membrane guanylate cyclase, and increased cGMP production in mixed bone cells. However, C-type natriuretic peptide did not increase cGMP in purified osteoclasts. By contrast, osteoclasts did produce cGMP in response to nitric oxide (NO) generators, sodium nitroprusside or 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazene. These findings indicate that C-type natriuretic peptide and NO modulate cGMP in different types of bone cells. The activity of the osteoclast centers on HCI secretion that dissolves bone mineral, and both NO generators and hydrolysis-resistant cGMP analogues reduced bone degradation, while cGMP antagonists increased activity. NO synthase agonists did not affect activity, arguing against autocrine NO production. Osteoclasts express NO-activated guanylate cyclase and cGMP-dependent protein kinase (G-kinase). G-kinase reduced membrane HCI transport activity in a concentration-dependent manner, and phosphorylated a 60-kD osteoclast membrane protein, which immunoprecipitation showed is not an H+-ATPase subunit. We conclude that cGMP is a negative regulator of osteoclast activity. cGMP is produced in response to NO made by other cells, but not in response to C-type natriuretic peptide. G-kinase modulates osteoclast membrane HCI transport via intermediate protein(s) and may mediate cGMP effects in osteoclasts.     

Non-enzymatic glycation of bone collagen modifies osteoclastic activity and differentiation

Type I collagen, the major organic component of bone matrix, undergoes a series of post-translational modifications that occur with aging, such as the non-enzymatic glycation. This spontaneous reaction leads to the formation of advanced glycation end products (AGEs), which accumulate in bone tissue and affect its structural and mechanical properties. We have investigated the role of matrix AGEs on bone resorption mediated by mature osteoclasts and the effects of exogenous AGEs on osteoclastogenesis. Using in vitro resorption assays performed on control- and AGE-modified bone and ivory slices, we showed that the resorption process was markedly inhibited when mature osteoclasts were seeded on slices containing matrix pentosidine, a well characterized AGE. More specifically, the total area resorbed per slice, and the area degraded per resorption lacuna created by osteoclasts, were significantly decreased in AGE-containing slices. This inhibition of bone resorption was confirmed by a marked reduction of the release of type I collagen fragments generated by the collagenolytic enzymes secreted by osteoclasts in the culture medium of AGE-modified mineralized matrices. This effect is likely to result from decreased solubility of collagen molecules in the presence of AGEs, as documented by the reduction of pepsin-mediated digestion of AGE-containing collagen. We found that AGE-modified BSA totally inhibited osteoclastogenesis in vitro, most likely by impairing the commitment of osteoclast progenitors into pre-osteoclastic cells. Although the mechanisms remain unknown, AGEs might interfere with osteoclastic differentiation and activity through their interaction with specific cell-surface receptors, because we showed that both osteoclast progenitors and mature osteoclasts expressed different AGEs receptors, including receptor for AGEs (RAGEs). These results suggest that AGEs decreased osteoclast-induced bone resorption, by altering not only the structural integrity of bone matrix proteins but also the osteoclastic differentiation process. We suggest that AGEs may play a role in the alterations of bone remodeling associated with aging and diabetes.     

Acidosis, hypoxia and bone

Bone homeostasis is profoundly affected by local pH and oxygen tension. It has long been recognised that the skeleton contains a large reserve of alkaline mineral (hydroxyapatite), which is ultimately available to neutralise metabolic H⁺ if acid-base balance is not maintained within narrow limits. Bone cells are extremely sensitive to the direct effects of pH: acidosis inhibits mineral deposition by osteoblasts but it activates osteoclasts to resorb bone and other mineralised tissues. These reciprocal responses act to maximise the availability of OH⁻ ions from hydroxyapatite in solution, where they can buffer excess H⁺. The mechanisms by which bone cells sense small pH changes are likely to be complex, involving ion channels and receptors in the cell membrane, as well as direct intracellular effects. The importance of oxygen tension in the skeleton has also long been known. Recent work shows that hypoxia blocks the growth and differentiation of osteoblasts (and thus bone formation), whilst strongly stimulating osteoclast formation (and thus bone resorption). Surprisingly, the resorptive function of osteoclasts is unimpaired in hypoxia. In vivo, tissue hypoxia is usually accompanied by acidosis due to reduced vascular perfusion and increased glycolytic metabolism. Thus, disruption of the blood supply can engender a multiple negative impact on bone via the direct actions of reduced pO₂ and pH on bone cells. These observations may contribute to our understanding of the bone disturbances that occur in numerous settings, including ageing, inflammation, fractures, tumours, anaemias, kidney disease, diabetes, respiratory disease and smoking.     

Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption

During skeletal growth and remodeling the mineralized bone matrix is resorbed by osteoclasts through the constant secretion of protons and proteases to the bone surface. This relies on the formation of specialized plasma membrane domains, the sealing zone and the ruffled border, and vectorial transportation of intracellular vesicles in bone-resorbing osteoclasts. Here we show that Rab7, a small GTPase that is associated with late endosomes, is highly expressed and is predominantly localized at the ruffled border in bone-resorbing osteoclasts. The decreased expression of Rab7 in cultured osteoclasts by antisense oligodeoxynucleotides disrupted the polarization of the osteoclasts and the targeting of vesicles to the ruffled border. These impairments caused a significant inhibition of bone resorption in vitro. The results indicate that the late endocytotic pathway is involved in the osteoclast polarization and bone resorption and underscore the importance of Rab7 in osteoclast function.     

Cytoplasmic pH is regulated in isolated avian osteoclasts by a Cl-/HCO3- exchanger

Osteoclast resorb bone in an acid compartment formed by the bone-attachment site. The low pH of the resorption compartment provides a lysosome-like milieu suitable for acid proteases to degrade collagen. Solubilization of the hydroxyapatite that makes up bone mineral consumes about 2 moles of protons per moles of calcium dissolved, requiring a massive proton flux to maintain a low pH in the resorption compartment. In order to determine how the osteoclast maintains a physiological cytoplasmic pH while secreting massive amounts of acid, we studied the intracellular pH of osteoclasts using esterified fluorescein derivatives while controlling the electrolyte composition of the medium. The principal finding is that osteoclasts have a high capacity for chloride/bicarbonate exchange which enables them to maintain normal intracellular pH in the face of a large loading of base equivalents. Thus, the overall process of proton secretion during bone resorption is similar to the polarized acid elimination by renal epithelia, involving a proton pump on one surface of the cell, and a Cl-/HCO3- exchange to maintain cytoplasmic pH.     

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.
1. The resorption of bone is the result of successive steps that can be regulated individually.
2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone.
3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation.
4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells.
5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO, by carbonic anhydrase and secreted by an ATP-driven vacuolar H⁺-K⁺-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone.
6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca²⁺) generated as a result of osteoclastic bone resorption.
7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.     

Osteoclast-independent bone resorption by fibroblast-like cells

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.     

Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis

Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, β-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.     

ATPase pumps in osteoclasts and osteoblasts

Osteoblasts, osteocytes and osteoclasts are specialised cells of bone that play crucial roles in the formation, maintenance and resorption of bone matrix. Bone formation and resorption critically depend on optimal intracellular calcium and phosphate homeostasis and on the expression and activity of plasma membrane transport systems in all three cell types. Osteotropic agents, mechanical stimulation and intracellular pH are important parameters that determine the fate of bone matrix and influence the activity, expression, regulation and cell surface abundance of plasma membrane transport systems. In this paper the role of ATPase pumps is reviewed in the context of their expression in bone cells, their contribution to ion homeostasis and their relation to other transport systems regulating bone turnover.     

Rab13 is upregulated during osteoclast differentiation and associates with small vesicles revealing polarized distribution in resorbing cells

Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts.     

Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid- serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.