Original Full Length ArticleA specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts
Highlights
► Only mature osteoclasts are sources of anabolic stimuli for osteoblasts. ► Osteopetrotic osteoclasts secrete anabolic factors for osteoblasts. ► Enhanced osteoclast survival compensates for the lack of resorption derived factors. ► Osteoclasts on non-natural substrates fail to secrete anabolic molecules. ► Interaction with matrix strongly modulates anabolic potential of the osteoclasts.
Introduction
Bone remodeling is an essential process for maintaining bone strength. Bone remodeling is a complex process most likely initiated by the osteocytes sensing stress, which then activate the osteoclasts leading to bone resorption [1], [2]. After bone resorption bone formation by the osteoblasts is initiated and the removed bone is completely replaced, a process originally identified and termed coupling by Harold Frost and co-workers [1], [3], [4], [5], [6]. Further evidence that coupling was to some extent controlled locally in bone was published by the Baylink group, who showed that bone organ cultures with activated resorption, secreted a bone anabolic molecule [7].
Alterations in the balance between bone resorption and bone formation result in pathologies, of which the most common is osteoporosis, where the osteoblasts are unable to counter the excessive bone resorption taking place [8]. Another situation, where the balance is destroyed, is the much rarer cases of osteoclast-rich forms of osteopetrosis [9]. In these cases bone resorption is strongly reduced, often due to defective acid secretion by the osteoclasts, while osteoclast numbers are increased [9]. Importantly, bone formation by the osteoblasts is often increased [8]. Furthermore, it was shown that the number of mature bone forming osteoblasts in these patients correlated to the number of non-resorbing osteoclasts [10], [11]. These findings highlight that anabolic signaling from the osteoclasts is not strictly mediated by bone resorption, but most likely also involves the presence of osteoclasts [8]. Additionally, a study conducted in adult mice indicated that when the mature non-resorbing osteoclasts are present, this leads to increased bone formation resulting in stronger bones [12]. Furthermore, studies conducted in c-fos deficient mice, which have macrophages but no osteoclasts, showed reduced bone formation, as well as failure to provide an anabolic response to PTH, while c-src deficient mice, which have non-resorbing osteoclasts, showed a full anabolic response to PTH treatment [13], [14], [15]. Finally, these data are supported by early findings in humans with no osteoclasts, where the bone phenotype is milder than that observed in osteoclast-rich osteopetrosis, despite both phenotypes having no bone resorption [16], [17].
Within recent years, several studies have highlighted that an osteoclasts are different depending on the circumstances, a phenomenon termed osteoclast subtypes [8]. These studies have indicated that resorptive activity, acid transport and proteolytic machinery and more parameters are highly influenced by the origin of the osteoclast as well as the interaction with the matrix [18], [19], [20], [21], [22], [23], [24], [25], [26], [27]. However, how the differences in osteoclast subtype influence the anabolic activity of the osteoclasts is presently unclear.
Following the paradigm shift in the understanding of the coupling between osteoclasts and osteoblasts, several studies have studied local communication between osteoclasts and osteoblasts, and how different parameters, such as bone resorption and osteoclast subtype, regulate this process [8]. Several studies have indicated different molecular candidates, including TGF-β, the IGFs, Wnt10b, BMP-6, S1P, TRACP, Cardiotrophin-1 and bidirectional signaling through the ephrin-eph system, and several of these are independent of bone resorption [6], [28], [29], [30].
When it comes to how much of the coupling signal that is derived from osteoclasts and how much that originates from bone resorption, this is still unclear. However, it was shown that cathepsin K inhibitors lead to increased secretion of bone matrix derived anabolic molecules, such as IGF-1, as the inhibitors prevented degradation of these factors [31], although it presently is unclear whether this has any relevance in vivo [32], [33].
With these things in mind, this study focused on providing a thorough investigation of when the osteoclasts secrete anabolic molecules. Importantly, we utilized naturally non-resorbing osteoclasts, as well resorption inhibitors to investigate the role resorption in this process. Furthermore, we investigated whether seeding the osteoclasts on different matrices, including the non-remodeled matrix dentine and the “artificial” matrix decalcified bone, modulated the ability of the osteoclasts to secrete anabolic molecules.
Section snippets
Chemicals and other reagents
Diphyllin was from Bioduro, Beijing, China, GM6001 and E64 from Calbiochem. All other materials were from Sigma-Aldrich unless otherwise specified.
CD34+ Cell isolation
Samples of peripheral blood from an Infantile Malignant Osteopetrosis patient or umbilical cord blood from normal deliveries were obtained after informed consent under protocols approved by institutional ethical boards. Mononuclear cells from these cell sources were isolated on Ficoll gradient, and subsequently CD34+ cells were separated from the
The appearance of the bone anabolic signal correlates with the appearance of osteoclasts
Previous studies have shown that osteoclasts secrete anabolic signals independent of bone resorption [29], [30]; however, at what time of development and whether macrophages possess this ability when cultured long term is not known. To shed light on the time profile of secretion of anabolic molecules by osteoclasts and the corresponding macrophages, we cultured CD14+ monocytes on bone slices in the presence or absence of RANKL, while we collected the conditioned media (CM) and the corresponding
Discussion
In this study we investigated the subtype of osteoclast needed from communication from osteoclasts to osteoblasts in detail, and we here show that osteoclasts, both dependently and independently of bone resorption produce bone anabolic signals to osteoblasts. Furthermore, our data show that the interaction between osteoclasts and culture matrix is highly important, as matrices such as dentine and decalcified bone attenuated the ability of the osteoclasts to produce anabolic signals.
These
Author contributions
KH and AVN devised most of the experiments and assisted in data analysis. KH wrote the manuscript. KVA and CECH performed the majority of the experiments and analyzed data. CST and NSG performed the CD34+ osteoclast experiments. JH, IM, AS provided and expanded the CD34+ cells from both IMO and control. MHD provided human blood. MAK participated in data analysis.
All authors have read, commented and approved the manuscript.
Conflicts of Interest
Morten A. Karsdal owns stock options in Nordic Bioscience A/S, all other authors have no conflicts of interest.
Acknowledgments
We thank the Danish Research Foundation (Den Danske Forskningsfond) for support of Kim Henriksen and Kim V. Andreassen. The support from the Ministry of Science, Technology and Innovation for the PhD education of the PhD education of Anita V. Neutzsky-Wulff and Catherine E. Crüger-Hansen is acknowledged. Christian S. Thudium is on a PhD Scholarship partially funded by Nordforsk. IM received funding by Blanceflor. JR was supported by grants from The Swedish Childhood Cancer Foundation, a
References (63)
- et al.
Local communication on and within bone controls bone remodeling
Bone
(2009) - et al.
Osteoclast-derived activity in the coupling of bone formation to resorption
Trends Mol Med
(2005) - et al.
Are osteoclasts needed for the bone anabolic response to parathyroid hormone? A study of intermittent parathyroid hormone with denosumab or alendronate in knock-in mice expressing humanized RANKL
J Biol Chem
(2010) - et al.
Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations
Am J Hum Genet
(2008) - et al.
Osteoclast heterogeneity: lessons from osteopetrosis and inflammatory conditions
Biochim Biophys Acta
(2009) - et al.
Proteolytic processing and polarized secretion of tartrate-resistant acid phosphatase is altered in a subpopulation of metaphyseal osteoclasts in cathepsin K-deficient mice
Bone
(2007) - et al.
Jaw and long bone marrow derived osteoclasts differ in shape and their response to bone and dentin
Biochem Biophys Res Commun
(2011) - et al.
Osteoclast polarization is not required for degradation of bone matrix in rachitic FGF23 transgenic mice
Bone
(2008) - et al.
Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis
Cell Metab
(2006) - et al.
Osteoclasts secrete non-bone derived signals that induce bone formation
Biochem Biophys Res Commun
(2008)
Cathepsin K inhibitors prevent matrix-derived growth factor degradation by human osteoclasts
Bone
Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression
J Biol Chem
Characterization of acid flux in osteoclasts from patients harboring a G215R mutation in ClC-7
Biochem Biophys Res Commun
Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II
Am J Pathol
High peripheral blood progenitor cell counts enable autologous backup before stem cell transplantation for malignant infantile osteopetrosis
Biol Blood Marrow Transplant
Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis
Am J Pathol
Structural and histomorphometric studies of iliac crest trabecular and cortical bone in autosomal dominant osteopetrosis: a study of two radiological types
Bone
Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorption
Am J Pathol
A physical mechanism for coupling bone resorption and formation in adult human bone
Am J Pathol
Osteal macrophages: a new twist on coupling during bone dynamics
Bone
Dissolution of the inorganic phase of bone leading to release of calcium regulates osteoclast survival
Biochem Biophys Res Commun
Bone quality—the material and structural basis of bone strength and fragility
N Engl J Med
Resorption precedes formative activity
Surg Forum
Suggested sequential mode of control of changes in cell behaviour in adult bone remodelling
Nature
Are nonresorbing osteoclasts sources of bone anabolic activity?
J Bone Miner Res
Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism
Proc Natl Acad Sci U S A
Osteoclast activity and subtypes as a function of physiology and pathology–implications for future treatments of osteoporosis
Endocr Rev
Advances in osteoclast biology resulting from the study of osteopetrotic mutations
Hum Genet
Osteoclast-derived serum tartrate-resistant acid phosphatase 5b in Albers–Schonberg disease (type II autosomal dominant osteopetrosis)
Clin Chem
Genetics, pathogenesis and complications of osteopetrosis
Bone
Dissociation of bone resorption and bone formation in adult mice with a non-functional V-ATPase in osteoclasts leads to increased bone strength
PLoS One
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