The roles of bone‐derived exosomes and exosomal microRNAs in regulating bone remodelling

Abstract Pathological destructive bone diseases are primarily caused by the failure of a lifelong self‐renewal process of the skeletal system called bone remodelling. The mechanisms underlying this process include enhanced osteoclast activity and decreased generation of the osteoblast lineage. Intercellular interaction and crosstalk among these cell types are crucial for the maintenance of bone remodelling, either through the secretion of growth factors or direct cell–cell physical engagement. Recent studies have revealed that exosomes derived from bone cells, including osteoclasts, osteoblasts and their precursors, play pivotal roles on bone remodelling by transferring biologically active molecules to target cells, especially in the processes of osteoclast and osteoblast differentiation. Here, we review the contents of bone‐derived exosomes and their functions in the regulatory processes of differentiation and communication of osteoclasts and osteoblasts. In addition, we highlight the characteristics of microRNAs of bone‐derived exosomes involved in the regulation of bone remodelling, as well as the potential clinical applications of bone‐derived exosomes in bone remodelling disorders.


Introduction
The characteristics and contents of bone-derived exosomes The roles of bone-derived exosomes in bone remodelling Conclusions and perspective Introduction Pathological destructive bone diseases [1], including osteoporosis, osteoarthritis (OA) and rheumatoid arthritis (RA) [2], are associated with a persistent decrease in the patient's quality of life [3] and are also considered to present a major global health problem [4]. These disorders are primarily caused by the failure of bone remodelling processes, including enhanced osteoclast activity or decreased generation of the osteoblast lineage [1].
Bone remodelling, which takes place in the bone remodelling compartment, is a continuous and lifelong process of repair of micro-damage to the bone structure and the replacement of ageing bone tissue [5]. It is a prerequisite for the maintenance of bone mass and the mechanical strength of bone [6]. In bone remodelling, the destructive process that involves resorption of bone by osteoclasts is coupled with a productive process, in which bone is synthesized by osteoblasts [7]. Osteoclasts, which share precursors with macrophages, are derived from haematopoietic stem cells (HSCs) [8] and are unique in their function of the resorption of bone matrices [9]. In contrast, along with adipocytes and chondrocytes, osteoblasts and their critical molecules required for the differentiation and communication of osteoclasts and osteoblasts [32].
The contents of exosomes vary according to their origin. To provide a better understanding of the mechanisms by which bonederived exosomes regulate bone remodelling, we have reviewed the latest discoveries regarding the changing characteristics and roles of bone-derived exosomes during the regulation of osteoclast and osteoblast differentiation. Furthermore, we highlight the characteristics of bone-derived exosomal microRNAs involved in regulation of bone remodelling as well as the potential clinical applications of bone-derived exosomes in bone remodelling disorders.

The characteristics and contents of bone-derived exosomes
Although the processes by which bioactive molecules are packaged into exosomes are largely unknown, recent reports suggest the existence of a specific and tightly controlled mechanism [33]. Isolation of exosomes from mature osteoclasts and precursors revealed they had similar sizes and morphology, as well as expression of specific markers, including epithelial cell adhesion molecule [34] and CD63 [35]. Sun et al. [32] indicated that ephrinA2 protein was enriched in osteoclast-derived exosomes. Furthermore, ephrinA2 levels in the serum of osteoporotic mice and patients were found to be significantly upregulated. These results suggested that osteoclast-derived exosomal ephrinA2 is a marker for recognition with osteoblasts, and the engagement of ephrinA2/EphA2 is essential for exosome-mediated communication between osteoclasts and osteoblasts. Receptor activator of nuclear factor jB (RANK) was detected at low levels in the exosomes from precursors but at much higher levels in mature osteoclasts [20]. In accordance with the induction of RANKL by bone resorption factors, such as parathyroid hormone (PTH), RANKL expression increased significantly in exosomes secreted from PTHtreated osteoblasts [22]. In addition, PTH caused osteoblasts to release exosomes containing osteoblast membrane proteins and the exosome marker flotillin-2 [36]. Proteomic analysis of osteoblastderived exosomes [37] revealed that the exosomal proteins are predominantly involved in protein localization and intracellular signalling cascade and are mainly located in the plasma membrane and cytosol, with molecular functions focused on nucleotide binding and structural molecule activity. Furthermore, exosomal proteins were enriched among the eukaryotic initiation factor (EIF)2, protein ubiquitination and integrin signalling pathways. In total, 23 proteins, including EIF family members, PP1C and PABP, were the mapped to EIF2 signalling pathway. Osteoblasts were derived from MSCs, thus implicating putative roles of MSC-derived exosomes in the regulation of bone remodelling. Lai et al. [38] identified 857 exosomal proteins within MSCderived embryonic stem cell lines. Mesenchymal stem cells-derived exosomes expressed the characteristic markers, CD13, CD29, CD44, CD73 and CD105 [39]. Among the 1069 proteins identified in exosomes isolated from the MSC-derived osteoblast precursor MC3T3 cell line, 786 proteins are present in the ExoCarta database [37]. Seven messenger RNA (mRNAs), ACIN1, DDX6, DGKA, DKK2, Lsm2, RPS2 and Xsox17, showed significant differential expression over time in exosomes from differentiated human bone marrow-derived mesenchymal stem cells (HBMSCs), which can be induced to differentiate into mineralized osteoblasts. Furthermore, dysregulated exosomal expression of two NF-jB-related genes, ADAM17 and NF-jB1, was detected in osteogenic differentiated HBMSCs [40]. Differential expression of some miRNAs was also detected in the exosomes of mineralizing osteoblasts and HBMSCs, including some that have been confirmed to be functionally associated with bone remodelling [40,41].

The roles of bone-derived exosomes in bone remodelling
Similar to the function of cytokines and soluble factors, bone-derived exosomes can recruit BM-derived cells, such as HSCs (precursors of osteoclasts) and MSCs (precursors of osteoblasts), to the bone surface [25]. Osteoclast precursor-derived exosomes stimulated the formation of significantly greater numbers of osteoclasts compared with the numbers formed in the absence of exosomes. In contrast, significantly fewer osteoclasts were formed in the presence of osteoclast-derived exosomes compared with the numbers determined in 1,25-(OH)2D3-stimulated mouse marrow. RANK levels were much higher in osteoclast-derived exosomes, and the removal of exosomes containing RANK significantly alleviated the inhibition of osteoclastogenesis [20]. Therefore, RANK-rich exosomes may function as novel inhibitors by binding competitively to RANKL, thus preventing stimulation of the RANK signalling pathway in osteoclasts [42]. Furthermore, there was no significant change in differentiation of mouse BM haematopoietic precursors stimulated with recombinant RANKL and M-CSF to mature osteoclasts following their exposure to exosomes isolated from osteoclast precursors and osteoclasts [20]. These data imply that the inhibitory function of RANK-rich exosomes on osteoclastogenesis may be alleviated in the presence of high levels of RANKL.
The roles of exosomes in osteoclasts may provide clues as to how bone formation and absorption are orchestrated [20]. Exosomes from BM stromal cells (BMSCs) can also be involved in bone remodelling by directly regulating osteoblast proliferation and activity [43]. Mesenchymal stem cells -derived exosomes have been shown to upregulate expression of the growth factors, bone morphogenetic protein 9 (BMP9) and transforming growth factor-b1(TGF-b1) [44], both of which effectively induce the osteogenic differentiation of MSCs [45]. BMP9 induces osteogenic differentiation with greater potency than BMP2 [46]. Mesenchymal stem cells -derived exosomes bind and tether extracellular matrix (ECM) proteins, such as type I collagen and fibronectin, to the bone surface and biomaterials. This function also allows the use of MSC-derived exosomes as biomimetic tools that induce the differentiation of BMSCs into an osteogenic lineage [47]. In addition, exosomes are released by the osteoblast itself, thus establishing a positive feedback mechanism that promotes bone growth. Exosomes from the mineralizing MC3T3-E1 (mature osteoblast cell line) promoted osteogenic differentiation of the ST2 osteoblast precursor cell line, manifested by the up-regulated expression of osteogenic markers, runt-related transcription factor 2 (RUNX2) and alkaline phosphatase, as well as enhanced matrix mineralization [41]. Furthermore, EIF2 in osteoblast-derived exosomes may also induce MSCs to differentiate into osteoblasts [37]. Bone remodelling is known to be closely regulated by the interaction between osteoblasts and osteoclasts. In fact, osteoblast-and osteocyte-derived lysosomal membrane protein 1 (LAMP1)-positive exosomes also contain tartrate-resistant acid phosphatase, RANKL and osteoprotegerin (OPG), which are critical to osteoclast differentiation [48]. As Deng et al. [22] identified, RANKL-rich exosomes released from osteoblasts stimulated osteoclast formation. In addition, Omar et al. and Ekstrom et al. indicated that exosomes secreted from monocytes, which are derived from HSCs and share precursors with osteoclasts, stimulated the osteogenic differentiation of MSCs [49,50]. The recent studies of Li et al. and Sun et al. demonstrated a novel models of inhibitory effect on osteogenic differentiation by osteoclast-derived miR-214-containing exosomes [32,51]. Although the differentiation of osteoclasts and osteoblasts is under the regulation of bone-derived exosomes (Fig. 1), the cell type from which the most potent regulatory exosomes are derived and the mechanism by which the exosomes mediate bone remodelling remain to be investigated [42].
Matrix vesicles (MVs), known to be important in the development of vertebrate mineralizing tissue, share structures that are homologous to those that anchor exosomes to the ECM as well as similarities in morphological appearance and functional activities [52]. Scanning electron microscope (SEM) observations showed that MVs are either scattered among collagen fibres, or aggregated on the cell surface [52]. Recent studies suggest that autophagosomes containing mineral nuclei are released from the cell and transported to the plasma membrane as mineralizing exosomes [53]. This process implicates autophagic activity in endosomal processing and indicates that mineralization starts within vacuoles before export from the cell. Thus, mineralization, nucleation and crystal growth are likely to be regulated by bone-derived exosomes [52].

The involvement of bone-derived exosomal miRNAs in regulating bone remodelling
The function of exosomal miRNAs has become a focus of research because of their pivotal role in gene expression regulation. micro-RNAs are small endogenous non-coding RNA molecules, containing approximately 22 nucleotides. They are well known as post-transcriptional regulators of messenger RNA (mRNA) expression [54]. Within the cytoplasm, the enzyme known as Dicer converts pre-miRNA into miRNA [55], which is loaded into the RNA-induced silencing complex (RISC) [56]. The RISC binds to the seed region of the target mRNA [57]. Although the binding complementarity is usually imperfect, silencing of target mRNA by the associated miRNAs impacts on between 1% and 4% of human gene expression [58]. After packaging into exosomes, miRNAs and the RISC are transported through the interstitium or even to the peripheral blood [59]. Exosomal miRNAs are more resistant to ubiquitous RNases, extreme temperatures and pH levels, and prolonged storage in the extracellular environment. In addition, plasma membrane fusion and endocytosis are the currently identified mechanisms that account for the uptake of exosomal miR-NAs by their target cells, where they exert their biological functions [59].
As communicators in bone remodelling, exosomes derived from the osteoblast lineage contain miRNAs that target key osteoclast differentiation factors. miR-503-3p from mineralized osteoblast-derived exosomes may inhibit RANKL-induced osteoclast differentiation by regulating RANK expression [75]. In contrast, miR-148a, which is known to promote osteoclastogenesis by targeting the MAFB gene [76,77], was found to be up-regulated in HBMSC exosomes. In addition, Li, et al. showed that elevated serum exosomal miR-214-3p is associated with reduced bone formation in both elderly women and ovariectomized (OVX) mice [51]. Serum exosomal miR-214 levels were then found to be significantly elevated in osteoclast-specific miR-214 transgenic mice. The miR-214 enriched in osteoclastderived exosomes can be transferred into osteoblasts to inhibit their activity via ephrinA2/EphA2 [32]. A previous study by Wang et al. [78] identified that miR-214 inhibits osteoblast function by targeting ATF4, while their further experiments identified that miR-214 promotes osteoclastogenesis through PI3K/Akt pathway by targeting the Pten tensin homologue [79]. Therefore, miR-214-containing exosomes from osteoclasts may have multiple roles that favour bone destruction. Future investigations are required to clarify the potential functions of these exosome-associated miRNAs in regulating bone remodelling via mechanisms such as paracrine/autocrine signalling or in communication between osteoclasts and osteoblasts or with other cell types [40].

Conclusions and perspective
Here, a new aspect of the roles of bone-derived exosomes in bone remodelling has been elucidated. The content of bone-derived exosomes, including the proteins, mRNAs and miRNAs, varies according to different parent cells. Therefore, bone-derived exosomes exert multiple roles in bone remodelling (Fig. 1). Exosomes originating from osteoclast precursors activate differentiation of osteoclasts and osteoblasts. In contrast, RANK-containing exosomes from osteoclasts inhibit osteoclast formation. Osteoclast-derived exosomes also inhibit osteoblast formation or might promote osteoclastogenesis in terms of the high expression of miR-214. Furthermore, exosomes isolated from osteoblast precursors promote the formation and activities of osteoblasts, while exosomes derived from mature osteoblasts either induce the differentiation of MSCs into mineralizing osteoblasts and osteocytes or establish positive feedback among osteoblasts themselves. In addition, osteoblast-derived exosomes containing OPG may inhibit the osteoclast differentiation. Thus, osteoblastderived exosomes may exert antagonistic or synergistic activities on osteoclasts through transporting their contents. We can imply and then infer that the exosomes from mineralizing osteoblasts, as well as precursors of osteoclasts and osteoblasts are principally involved in promoting bone remodelling by facilitating either osteogenesis or osteoclastogenesis. However, the mature osteoclast-derived exosomes are likely to be inhibitory factors for bone remodelling through alleviating osteoclast differentiation and osteoblast formation.
Bone-derived exosomal miRNAs are thought to be important in regulating gene expression involved in the differentiation and communication between multiple cell types responsible for bone formation and resorption. Recent studies have shown that Wnt5a enhances osteoclast formation through b-catenin-dependent signalling [80]. RUNX2 is a key transcription factor that regulates osteogenesis, while osteocytes secrete sclerostin (encoded by the SOST gene) to inhibit the activity of osteoclasts and osteoblasts [81]. According to the literature, many of the key factors that regulate osteoclasts and osteoblasts are targeted by the different miRNAs contained within specific bone-derived exosomes, such as RUNX2, BMPs and sclerostin [42]. However, exosomal miRNAs from the same parent cells may have opposing functions in terms of osteoclast differentiation and osteoblast activity. Thus, this implies that bone-derived exosomes may not perform functions such as cell-cell interaction during the regulation of bone remodelling that are completely in accordance with those of the parent cells. microRNAs alterations in the recipient do not match the abundance of miRNA in the donor exosomes [41], suggesting that components of osteoblast-derived exosomes other than miRNAs may also alter the miRNA profile of recipient cells [41]. The endosomal sorting complexes required for transport (ESCRT) machinery functions as a link between miRNAs and exosomes. In this system, the RISC acts together with the ESCRT complex to load miRNAs into exosomes [59]. miRNAs form an integral part of the RISC complex, which mediates the efficient transfer of the miRNA to its target in the recipient cell. Furthermore, exosomes incorporate precursor miRNAs (pre-miRNAs) in complexes with Dicer, TRBP and AGO2 proteins, to facilitate their processing in a cell-independent manner [82].
The cargo of tumour-derived exosomes vary according to the cancer types and tumour characteristics [83,84], and are more relevant in malignancy than benign bone diseases. Therefore, information on the crosstalk between cancer cells and the bone microenvironment through exosomes is not discussed in the present paper, even though it is important to consider its effect on bone homeostasis. In terms of the multiple and complex interactions between types of bone cells reviewed herein, further investigations are needed to fully elucidate the contents and potential modulatory roles of bone-derived exosomes in regulating bone remodelling, especially the comprehensive protein and miRNA contents of osteoclast-derived exosomes as well as which types of bone-derived exosomes play dominant roles in the regulation of bone remodelling and under what circumstances they would be activated. Encapsulation by the lipid bilayer of the exosomal membrane protects proteins and miRNAs from degradation [85] and they can be assayed in small peripheral blood samples [33]. Thus, the different expression level of specific contents of bone-derived exosomes might serve as a promising diagnostic and/or prognostic tool to detect early-stage bone disorders. Furthermore, engineered exosomes represent a promising system that can be used for the targeted delivery of RNAi molecules, while avoiding detection by the immune system [86]. In this way, the transfer of particular ESCRT machinery or miRNAs might facilitate the treatment of bone diseases that are related to aberrant bone remodelling, such as RA, OA and osteoporosis.