A comparative view on mechanisms and functions of skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function

Resorption and remodelling of skeletal tissues is required for development and growth, mechanical adaptation, repair, and mineral homeostasis of the vertebrate skeleton. Here we review for the first time the current knowledge about resorption and remodelling of the skeleton in teleost fish, the largest and most diverse group of extant vertebrates. Teleost species are increasingly used in aquaculture and as models in biomedical skeletal research. Thus, detailed knowledge is required to establish the differences and similarities between mammalian and teleost skeletal remodelling, and between distantly related species such as zebrafish (Danio rerio) and medaka (Oryzias latipes). The cellular mechanisms of differentiation and activation of osteoclasts and the functions of teleost skeletal remodelling are described. Several characteristics, related to skeletal remodelling, distinguish teleosts from mammals. These characteristics include (a) the absence of osteocytes in most species; (b) the absence of haematopoietic bone marrow tissue; (c) the abundance of small mononucleated osteoclasts performing non‐lacunar (smooth) bone resorption, in addition to or instead of multinucleated osteoclasts; and (d) a phosphorus‐ rather than calcium‐driven mineral homeostasis (mainly affecting the postcranial dermal skeleton). Furthermore, (e) skeletal resorption is often absent from particular sites, due to sparse or lacking endochondral ossification. Based on the mode of skeletal remodelling in early ontogeny of all teleosts and in later stages of development of teleosts with acellular bone we suggest a link between acellular bone and the predominance of mononucleated osteoclasts, on the one hand, and cellular bone and multinucleated osteoclasts on the other. The evolutionary origin of skeletal remodelling is discussed and whether mononucleated osteoclasts represent an ancestral type of resorbing cells. Revealing the differentiation and activation of teleost skeletal resorbing cells, in the absence of several factors that trigger mammalian osteoclast differentiation, is a current challenge. Understanding which characters of teleost bone remodelling are derived and which characters are conserved should enhance our understanding of the process in fish and may provide insights into alternative pathways of bone remodelling in mammals.     

Characterization of morphology and physiological actions of scale osteoclasts in the rainbow trout

The morphology of scale osteoclasts in rainbow trout Oncorhynchus mykiss was characterized by light and scanning electron microscopy, and the effects of oestradiol-17β-treatment and sexual maturation on scale osteoclast morphology were investigated. The cells associated with resorption cavities could be distinguished morphologically as two types: symmetrical, compact cells lacking or having only a few cell processes, termed type 1 cells, and asymmetrical cells covered with folds and having several cell processes, termed type 2 cells. In adult sexually maturing fish, where scale resorption was high, type 1 cells were predominant. In juveniles and spawned adults where scale resorption was assumed to be relatively low, mostly type 2 cells were present. Oestradiol 17-β-treatment of juvenile rainbow trout increased the osteoclast activity, but did not affect the osteoclast morphology. Using light microscopy, the majority of the cells observed in, and closely associated with, the resorption cavities were mononucleated in both maturing and spawned fish. Occasionally, bi- and multinucleated osteoclasts were observed in the maturing, but not in the spawned fish. Light microscopic enzyme-histochemistry showed that the majority of the mononucleated cells, as well as the bi- and multinucleated ones, were tartrate resistant acid phosphatase positive in both groups of fish, thus implying that both type 1 and type 2 cells were osteoclasts. It is thus apparent that scale resorption in rainbow trout is carried out by two morphologically distinct osteoclast populations, representing different stages of osteoclast activity and/or different stages of osteoclast differentiation.     

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.     

Growth requires bone resorption at particular skeletal elements in a teleost fish with acellular bone (Oreochromis niloticus, Teleostei: Cichlidae)

To date, little is known about bone resorption during skeletal development in teleostean fish with acellular bone. We report here about bone resorption with regard to growth in the tilapia Oreochromis niloticus. Nine skeletal elements obtained from growing juveniles were examined using histological and histochemical methods, and transmission electron microscopy (TEM). Tartrate-resistant acid phosphatase (TRAP) served as a marker for bone resorbing cells (osteoclasts), alkaline phosphatase (ALP) was used to identify osteoblasts, and alizarin staining indicated sites of bone formation. TRAP-activity was located at those skeletal elements where growth requires bone resorption, and at sites of cartilage degeneration. No TRAP-activity was found at those skeletal elements where resorption was not required for growth. The examination of the praeopercular shaft leads to a model of bone enlargement, including bone resorption by TRAP-positive cells located at the endosteal bone surface and bone formation by ALP-positive cells located at the periosteal bone surface. TRAP-positive cells were mononucleated and lacked a ruffled border. They appeared either as cell aggregates (resembling the shape of multinucleated giant cells) or as flat cells (resembling bone lining cells). Problems of osteoclast identification in bony fish are discussed.     

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.     

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.     

Genetic regulation of osteoclast development and function

Osteoclasts are the principal, if not exclusive, bone-resorbing cells, and their activity has a profound impact on skeletal health. So, disorders of skeletal insufficiency, such as osteoporosis, typically represent enhanced osteoclastic bone resorption relative to bone formation. Prevention of pathological bone loss therefore depends on an appreciation of the mechanisms by which osteoclasts differentiate from their precursors and degrade the skeleton. The past five years have witnessed important insights into osteoclast formation and function. Many of these discoveries have been made through genetic experiments that involved the rare hereditary disorder osteopetrosis.     

The osteoclast: a multinucleated, hematopoietic-origin, bone-resorbing osteoimmune cell

Osteoclasts are multinucleated cells that derive from hematopoietic progenitors in the bone marrow which also give rise to monocytes in peripheral blood, and to the various types of tissue macrophages. Osteoclasts are formed by the fusion of precursor cells. They function in bone resorption and are therefore critical for normal skeletal development (growth and modeling), for the maintenance of its integrity throughout life, and for calcium metabolism (remodeling). To resorb bone, the osteoclasts attach to the bone matrix, their cytoskeleton reorganizes, and they assume polarized morphology and form ruffled borders to secrete acid and collagenolytic enzymes and a sealing zone to isolate the resorption site. Identification of the osteoclastogenesis inducer, the receptor activator of nuclear factor-kappaB ligand (RANKL), its cognate receptor RANK, and its decoy receptor osteoprotegerin (OPG), has contributed enormously to the dramatic advance in our understanding of the molecular mechanisms involved in osteoclast differentiation and activity. This explosion in osteoclast biology is reflected by the large number of reviews which appeared during the last decade. Here I will summarize the "classical" issues (origin, differentiation, and activity) in a general manner, and will discuss an untouched issue (multinucleation) and a relatively novel aspect of osteoclast biology (osteoimmunology).     

MCP-1-induced human osteoclast-like cells are tartrate-resistant acid phosphatase, NFATc1, and calcitonin receptor-positive but require receptor activator of NFkappaB ligand for bone resorption

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFkappaB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP(+)/CTR(+) multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP(+)/CTR(+) multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP(+)/CTR(+) multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP(+)/CTR(+) multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.     

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.     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

TNAP, TrAP, ecto-purinergic signaling, and bone remodeling

Bone remodeling is a process of continuous resorption and formation/mineralization carried out by osteoclasts and osteoblasts, which, along with osteocytes, comprise the bone multicellular unit (BMU). A key component of the BMU is the bone remodeling compartment (BRC), isolated from the marrow by a canopy of osteoblast-like lining cells. Although much progress has been made regarding the cytokine-dependent and hormonal regulation of bone remodeling, less attention has been placed on the role of extracellular pH (pH(e)). Osteoclastic bone resorption occurs at acidic pH(e). Furthermore, osteoclasts can be regarded as epithelial-like cells, due to their polarized structure and ability to form a seal against bone, isolating the lacunar space. The major ecto-phosphatases of osteoclasts and osteoblasts, acid and alkaline phosphatases, both have ATPase activity with pH optima several units different from neutrality. Furthermore, osteoclasts and osteoblasts express plasma membrane purinergic P2 receptors that, upon activation by ATP, accelerate bone osteoclast resorption and impair osteoblast mineralization. We hypothesize that these ecto-phosphatases help regulate [ATP](e) and localized pH(e) at the sites of bone resorption and mineralization by pH-dependent ATP hydrolysis coupled with P2Y-dependent regulation of osteoclast and osteoblast function. Furthermore, osteoclast cellular HCO3(-), formed as a product of lacunar V-ATPase H(+) secretion, is secreted into the BRC, which could elevate BRC pH(e), in turn affecting osteoblast function. We will review the existing data addressing regulation of BRC pH(e), present a hypothesis regarding its regulation, and discuss the hypothesis in the context of the function of proteins that regulate pH(e).     

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.     

Biology of RANK,RANKL, and osteoprotegerin

The discovery of the receptor activator of nuclear factor-kappaB ligand (RANKL)/RANK/osteoprotegerin (OPG) system and its role in the regulation of bone resorption exemplifies how both serendipity and a logic-based approach can identify factors that regulate cell function. Before this discovery in the mid to late 1990s, it had long been recognized that osteoclast formation was regulated by factors expressed by osteoblast/stromal cells, but it had not been anticipated that members of the tumor necrosis factor superfamily of ligands and receptors would be involved or that the factors involved would have extensive functions beyond bone remodeling. RANKL/RANK signaling regulates the formation of multinucleated osteoclasts from their precursors as well as their activation and survival in normal bone remodeling and in a variety of pathologic conditions. OPG protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from binding to its receptor, RANK. Thus, RANKL/OPG ratio is an important determinant of bone mass and skeletal integrity. Genetic studies in mice indicate that RANKL/RANK signaling is also required for lymph node formation and mammary gland lactational hyperplasia, and that OPG also protects arteries from medial calcification. Thus, these tumor necrosis factor superfamily members have important functions outside bone. Although our understanding of the mechanisms whereby they regulate osteoclast formation has advanced rapidly during the past 10 years, many questions remain about their roles in health and disease. Here we review our current understanding of the role of the RANKL/RANK/OPG system in bone and other tissues.     

Identification of osteopontin in isolated rabbit osteoclasts.

Bone remodeling is a complex process coupling bone formation and resorption. Osteoblasts, the bone-forming cells, are known to produce various bone matrix proteins and cytokines; however, little is known about protein factors produced by osteoclasts or bone-resorbing cells. A method utilizing the high affinity of osteoclasts for tissue culture dishes was developed to isolate a large number of pure osteoclasts from rabbit long bones. A cDNA library was then constructed from these isolated osteoclasts, and differential cDNA screening was performed between osteoclasts and spleen cells. Two clones representing osteoclast-specific clones, named OC-1 and OC-2, were isolated. By Northern blot analysis, OC-1 was expressed in osteoclasts and in kidneys, whereas OC-2 was specific for osteoclasts. OC-1 was found to encode osteopontin from its nucleotide sequence, and therefore, osteopontin may have other functions for osteoclastic bone resorption besides osteoclast attachment to bone.     

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.     

Histone deacetylases in control of skeletogenesis

Skeletogenesis occurs continuously during the lifespan of vertebrate organisms. In development, the skeleton is patterned and modeled until each bone achieves its optimal shape and full size. During adults, the skeleton is remodeled to maintain strength and release calcium. The bone-resorbing and bone-forming activities of osteoclasts and osteoblasts, respectively, are tightly coupled to maintain optimal skeletal health; however, during aging and disease, these cells can become uncoupled, adversely affecting skeletal health and strength. Histone deacetylases have emerged as important regulators of endochondral bone formation, osteoblast maturation and osteoclast survival. Histone deacetylases are inhibited by small molecules that are approved and/or in clinical trials as cancer therapeutic drugs or anti-epileptic agents. In this article, the roles of histone deacetylases and effects of histone deacetylase inhibitors on bone and cartilage cells are reviewed.     

Bone morphogenetic protein-9 activates Smad and ERK pathways and supports human osteoclast function and survival in vitro

BMP-9 is a potent osteogenic factor; however, its effects on osteoclasts, the bone-resorbing cells, remain unknown. To determine the effects of BMP-9 on osteoclast formation, activity and survival, we used human cord blood monocytes as osteoclast precursors that form multinucleated osteoclasts in the presence of RANKL and M-CSF in long-term cultures. BMP-9 did not affect osteoclast formation, but adding BMP-9 at the end of the culture period significantly increased bone resorption compared to untreated cultures, and reduced both the rate of apoptosis and caspase-9 activity. BMP-9 also significantly downregulated the expression of pro-apoptotic Bid, but only after RANKL and M-CSF, which are both osteoclast survival factors, had been eliminated from the culture medium. To investigate the mechanisms involved in the effects of BMP-9, we first showed that osteoclasts expressed some BMP receptors, including BMPR-IA, BMPR-IB, ALK1, and BMPR-II. We also found that BMP-9 was able to induce the phosphorylation of Smad-1/5/8 and ERK 1/2 proteins, but did not induce p38 phosphorylation. Finally, knocking down the BMPR-II receptor abrogated the BMP-9-induced ERK-signaling, as well as the increase in bone resorption. In conclusion, these results show for the first time that BMP-9 directly affects human osteoclasts, enhancing bone resorption and protecting osteoclasts against apoptosis. BMP-9 signaling in human osteoclasts involves the canonical Smad-1/5/8 pathway, and the ERK pathway.