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Bone Cell Communication Factors Provide a New Therapeutic Strategy for Osteoporosis

Bone homeostasis is strictly regulated by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Many studies have shown that osteoclasts affect osteoblasts, and vice versa, through diffusible paracrine factors, cell-cell contact, and cell-bone matrix interactions to achieve the correct balance between osteoclastic and osteoblastic activities in the basic multicellular unit (BMU). The strict regulation that occurs during bone remodeling hinders the long-term use of the currently available antiresorptive agents and anabolic agents for the treatment of osteoporosis. To overcome these limitations, it is necessary to develop novel agents that simultaneously inhibit bone resorption, promote bone formation, and decouple resorption from formation. Therefore, a more detailed understanding of the mechanisms involved in osteoclast-osteoblast communication during bone remodeling is necessary.


INTRODUCTION
The bone is a dynamic organ that undergoes continuous renewal through bone remodeling processes to maintain its mechanical characteristics and calcium homeostasis. Bone remodeling is a complex and sophisticated series of sequential events, which occur within a temporary anatomical structure called the basic multicellular unit (BMU), involving various cell types including osteoclasts, osteoblasts, osteocytes, T-cells, macrophages, pericytes, vascular endothelial cells, canopy bone lining cells, and precursor populations of osteoblasts and osteoclasts. [1][2][3] In particular, osteoclasts and osteoblasts are the two major cells regulating bone remodeling processes. Osteoclasts and osteoblasts are responsible for old bone resorption and new bone formation, respectively. Bone remodeling in each BMU proceeds in cycles consisting of distinct phases: the recruitment of osteoclasts and bone resorption by osteoclasts; the coupling of resorption to formation or reversal from catabolism to anabolism; the recruitment of osteoblasts and new bone formation by osteoblasts; and the termination of these processes. [4][5][6][7] As an imbalance between bone formation and bone resorption results in multiple metabolic bone diseases like osteoporosis and osteopetrosis, each phase of the bone remodeling process must be strictly regulated by various local or systemic factors and intracellular signals to maintain bone homeostasis. [8][9][10][11] Osteoporosis is the most common metabolic bone disease caused by excessive bone resorption relative to formation. It is characterized by low bone mass, the deterioration of bone tissue, and an increased risk of bone fracture. Osteoporosis-related fractures most commonly occur in the hip, wrist, spine, or shoulder, particularly in post-menopausal women. [8][9][10][11] Several drugs are currently available for osteoporosis treatment. These drugs target either the inhibition of bone resorption or the promotion of bone formation. However, certain limitations of antiresorptive agents and boneforming drugs have been revealed. Antiresorptive drugs, such as alendronate, zoledronic acid, risedronate, and ibandronate, effectively block the formation and function of osteoclasts, but simultaneously reduce bone formation. In contrast, anabolic drugs, such as parathyroid hormones, teriparatide, and recombinant human parathyroid hormone, increase bone formation markers, but also increase bone resorption markers. These long-term adverse events induced by antiresorptives and bone-forming drugs suggest that the coupling process between bone resorption and formation plays a crucial role in the complete restoration of the bone removed during remodeling cycles. 8,[10][11][12][13][14] Therefore, an understanding of the signaling pathway involved in the coupling process will help develop novel drugs that simultaneously block bone resorption and promote bone formation without certain adverse events. Here, we have reviewed the coupling factors that may be an ideal target for the management of osteoporosis.

RANKL/RANK SIGNALING
The receptor activator of the nuclear factor kappa B ligand (RANKL) is an essential factor for osteoclast differentiation and function. It is secreted by osteoblasts and osteocytes, and binds to receptor activator of nuclear factor kappa B (RANK) on the surface of osteoclast precursors. 3,15,16 In addition, the physiological roles of the RANKL in osteoblasts have recently been elucidated. The vesicular RANK, secreted from maturing osteoclasts, binds to the osteoblastic RANKL to promote bone formation by osteoblasts. The osteoblastic RANKL regulates bone formation through the activation of PI3K-Akt mTOR to induce the expression of runt-related transcription factor 2 (Runx2). 3,17,18 Therefore, the RANKL-RANK system could regulate both bone resorption and bone formation by using RANKL forward signaling and RANKL reverse signaling, respectively.
Denosumab, a monoclonal antibody against RANKL, is available for the management of osteoporosis and skeletal problems caused by the spread of cancers to bone. Denosumab binds to RANKL, thereby inhibiting osteoclast forward signaling. Despite its efficacy in the inhibition of bone resorption, adverse effects, such as low bone formation, may impede long-term use. 8,[19][20][21] Interestingly, Ikebuchi and colleagues developed an anti-RANKL antibody that reduced osteoclast formation and function by binding and inactivating multiple RANKL monomers, and stimulated osteoblast differentiation by binding to the cell-surface of the RANKL. 17,18 Therefore, RANKL-RANK forward or reverse signaling offers a new strategy for the management of osteoporosis, which is able to trigger bone formation while inhibiting bone resorption.

SCLEROSTIN
Sclerostin is encoded by the SOST gene in humans. 22 After discovering that the lack of SOST expression was the cause of the high bone mass in human Van Buchem disease and sclerosteosis, considerable evidence from in vitro, animal, and human studies has demonstrated that sclerostin plays an important role in bone homeostasis. 23,24 Sclerostin is secreted primarily from osteocytes, but not osteoblasts. 23,25 It has been identified as binding to LRP5/6 receptors and antagonizing the canonical Wnt pathway. 26,27 The inhibition of the Wnt pathway by sclerostin leads to the inhibition of bone formation by osteoblasts. In addition, sclerostin stimulates bone resorption through its inhibitory action on the canonical Wnt pathway, because activation of the canonical Wnt pathway in osteoblasts increases the expression of osteoprotegrin (OPG), a decoy receptor for RANKL, and reduces bone resorption. 14,24,[28][29][30] Sclerostin expression is also detected in osteoclast precursors and its expression is decreased when osteoclasts are formed in vitro. 24,31 Tnfrsf11b(Opg)-/-and Tnfsf11 (Rankl)-transgenic mice with a high-bone turnover exhibited a low level of sclerostin, suggesting that the suppression of sclerostin was associated with bone resorption is critical for the coupling of bone resorption to formation. 27,32 Romosozumab, a monoclonal antibody against sclerostin, can simultaneously increase bone formation and decrease bone resorption when administered subcutaneously. However, it is usually administered for only 1 year owing to its gradual decrease in efficacy. 8,33-35

SLIT3
Slit guidance ligand (SLIT) proteins were originally identified as chemorepellents that controlled axon crossing in the midline of the brain. Recently, Kim et al. 36 reported that SLIT3 was a coupling factor to regulate resorption-formation coupling. SLIT3 production is increased during osteoclast differentiation. The secretion of SLIT3 by osteoclasts stimulates pre-osteoblast migration and β-cateninmediated osteoblast differentiation. In addition, SLIT3 suppresses osteoclast differentiation via the inhibition of Rac activation in an autocrine and paracrine manner. Therefore, the dual roles of SLIT3 in both osteoblasts and osteoclasts result in osteoporotic bone phenotypes that involve a decrease in bone formation and an increase of bone resorption in mice lacking Slit3 or its receptor Robo1. [36][37][38] Importantly, the injection of a truncated SLIT3 containing the ROBO-binding LRR2 domain into ovariectomized mice reversed ovariectomy-induced bone loss by simultaneously enhancing bone formation and reducing bone resorption. 3,36-38

SEMAPHORINS
Although semaphorins (SEMAs) were first identified as axon guidance cues, they have been shown to play important roles in angiogenesis, tissue development, and the immune response. [39][40][41][42] Of the eight classes of semaphorin family proteins, several studies have suggested important roles of SEMA4D and SEMA3A in bone metabolism. 3,43,44 SEMA4D is a transmembrane semaphorin highly expressed in osteoclasts, but not in osteoblasts. FC-SEMA4D, a soluble FC receptor SEMA4D fusion protein, inhibits osteoblast differentiation and function without altering proliferation. The binding of SEMA4D to its receptor complex, consisting of ErbB2 and Plexin-B1, leads to activation of the small GTPase RhoA. Genetically altered mice with Sema4d and Plxnb1 deletion, as well as mice expressing an osteoblast-targeted dominant-negative RhoA, exhibited a high bone mass due to enhanced osteoblastic bone formation. 45,46 However, the regulation of bone mass by SEMA4D may be more complicated. Dacquin et al. 44 reported that the increased bone mass phenotype in Sema4d-deficient mice was primarily due to a functional defect in osteoclasts. The authors showed that Sema4d-deficient primary osteoclasts led to delayed osteoclast differentiation and reduced osteoclast resorption activity that was in part due to the unbalanced regulation of β3 integrin subunit signaling. 44 Although the precise mechanisms through which SEMA4D contributes to bone homeostasis have not been elucidated, the injection of Sema4d siRNA or SEMA4Dspecific antibody into an ovariectomy-induced animal model of osteoporosis reversed bone mass, suggesting that SEMA4D was a beneficial target for osteoporosis treatment. 45,47 SEMA3A was first identified in the involvement of patterned neuronal connections and is now recognized as a mediator linking osteoclasts and osteoblasts. 48 SEMA3A is mainly expressed by osteoblasts and its receptor, Nrp1, is expressed by osteoclast precursors. [48][49][50] Sema3a-deficient osteoblasts showed a defect in osteoblast differentiation owing to the inhibition of β-catenin activation, whereas SEMA3A treatment caused a decrease in the differentiation of osteoclast precursors through the inhibition of RhoA activation. 51 Hayashi et al. 51 reported that a global Sema3a deletion in mice caused a severe osteopenic phenotype that was associated with a decrease in osteoblastic bone formation and an increase in osteoclastic bone resorption. Interestingly, mice with osteoblast-specific deletion of Sema3a did not undergo any change in bone parameters, whereas mice with neuron-specific deletion of Sema3a exhibited a markedly low bone mass, similar to mice with global deletion of Sema3a. 52 These results were indicative of the indirect effects of SEMA3A on bone metabolism through the nervous system. Furthermore, the injection of SEMA3A into ovariectomized mice prevented ovariectomyinduced bone loss, both through the promotion of bone formation and the suppression of bone resorption. 51

CTHRC1
Collagen triple helix repeat containing 1 (CTHRC1) was originally identified in injured arteries. 53 The expression of CTHRC1 was found to be induced in mature bone-resorbing osteoclasts. 54 The recombinant CTHRC1 protein stimulated osteoblastic differentiation of marrow stromal ST2 cells. Cthrc1 null mice showed a lower bone mass due to decreased bone formation, whereas Cthrc1 transgenic mice exhibited a higher bone mass owing to an increase in bone formation. 54 Collectively, evidence obtained from in vitro and in vivo experiments indicated that CTHRC1 was an important stimulator of osteoblastic bone formation. To further define whether CTHRC1 acted as a coupling factor, expressed only by mature bone-resorbing osteoclasts, to stimulate bone formation, recombinant RANKL was in-jected into mice with osteoclast-specific Cthrc1 deletion. The acute phase of osteoclastic bone resorption occurred to the same extent as in control mice, whereas the anabolic response followed by resorption was inhibited or delayed in the mice with osteoclast-specific deletion of Cthrc1. 54 In contrast, it has been shown that CTHRC1 was secreted by osteoblasts and some osteocytes, but not by osteoclasts. 55 In that study, the authors also demonstrated that CTHRC1 negatively regulated osteoclast differentiation through the inhibition of RANKL-induced NF-kB signaling activation and ERK1/2 phosphorylation. Their results revealed that the lower bone mass observed in Cthrc1-null mice was also the result of increased bone resorption as well as a decreased bone formation. Collectively, the in vitro and in vivo evidence supports the potential importance of CTHRC1 in bone remodeling; however, it remains to be determined if the role of CTHRC1 in bone remodeling is mediated by signals from the osteoblast lineage or from osteoclasts.

CONCLUSIONS
Generally, coupling factors are the molecules that are involved in the stimulation of osteoblastic bone formation in response to osteoclastic bone resorption to preserve normal bone mass. 3,56 However, recent studies have shown that some molecules, such as sclerostin, SEMA4D, and SEMA3A, control bone remodeling through cell-cell communication between bone cells rather than a classical coupling process. Negishi-Koga et al. 43,45 proposed that such factors should be called bone cell communication factors, as they participate in the bone remodeling process by regulating intercellular cross-talk among bone cells. 3 Herein, we have discussed bone cell communication factors that are likely to be ideal therapeutic targets for osteoporosis (Fig. 1). As the orchestration of bone remodeling is strictly regulated by various known and as yet unknown bone communication factors, future investigations should be focused on the discovery of additional coupling signals and elucidate how these factors coordinate resorption and formation coupling in concert.