Ubiquitin‐specific peptidases: Players in bone metabolism

Abstract Osteoporosis is an ageing‐related disease, that has become a major public health problem and its pathogenesis has not yet been fully elucidated. Substantial evidence suggests a strong link between overall age‐related disease progression and epigenetic modifications throughout the life cycle. As an important epigenetic modification, ubiquitination is extensively involved in various physiological processes, and its role in bone metabolism has attracted increasing attention. Ubiquitination can be reversed by deubiquitinases, which counteract protein ubiquitination degradation. As the largest and most structurally diverse cysteinase family of deubiquitinating enzymes, ubiquitin‐specific proteases (USPs), comprising the largest and most structurally diverse cysteine kinase family of deubiquitinating enzymes, have been found to be important players in maintaining the balance between bone formation and resorption. The aim of this review is to explore recent findings highlighting the regulatory functions of USPs in bone metabolism and provide insight into the molecular mechanisms governing their actions during bone loss. An in‐deep understanding of USPs‐mediated regulation of bone formation and bone resorption will provide a scientific rationale for the discovery and development of novel USP‐targeted therapeutic strategies for osteoporosis.

by antagonizing the osteoclast differentiation of bone marrow monocytes. [11][12][13][14] Therefore, USPs are potential new targets in bone metabolism-related disease mechanism research and drug development and show broad prospects for osteoporosis prevention and treatment. 13 In this review, we provide the latest knowledge on the impact of these USPs on bone metabolism, including their activity in mesenchymal stem cells (MSCs), osteoblasts, osteoclasts, and osteocytes (Table 1).

| USPs: STRUCTURAL AND FUNCTIONAL DOMAINS
USPs constitute a cysteine protease family and they range from 50 to 300 kDa, with enzymatic activity realized through the thiol group of the central cysteine. 41 The catalytic domain of a USP adopts a papainlike fold, and this conserved catalytic domain enables most USPs to hydrolyze isopeptide bonds via a similar mechanism. Different from other DUBs, USPs carry specific cysteine protease domains that exhibit functional catalytic mechanisms; these domains comprise three conserved subdomains that resemble the relationship between the palm, thumb and fingers of a right hand. 42 The catalytic site is located at the interface between the palm and thumb subdomains, while the finger subdomain is crucial for the interaction with a distal ubiquitin. 42 Most members of the USP family include a core catalytic domain and other N-and C-terminal extensions, and they may be composed of different domains or sequences with specific functions. 43 Ubiquitin is a small conserved regulatory protein comprising 76 amino acids and can be linked to methionine (M1) residues and lysine (K) residues, such as K6, K11, K27, K29, K33, K48 and K63. 44 Notably, K48 and K63 chains have been the most extensively studied ubiquitin chains, with the K48 linked ubiquitin chain found to be associated with ATP-dependent proteasome degradation, 45 while the K63 ubiquitin chain plays a role in signal transduction and DNA repair. 10,46 Most USPs show hydrolytic activity on K48-, K63-and Met1-linked ubiquitin chains and participate in the regulation of various biological processes, such as cell migration, tumorigenesis, immune responses, and inflammation. 44,47 The abnormal expression or mutation of USPs usually leads to a series of diseases 48 (Table 2), including bone metabolism disorders, such as osteoporosis. 3

| EFFECTS OF USPs ON THE SELF-RENEWAL AND OSTEOGENIC DIFFERENTIATION OF MSCs
Mesenchymal stem cells (MSCs) are multipotent progenitors with selfrenewal capability and multidirectional differentiation potential; that is, they can undergo osteogenesis, adipogenesis and chondrogenesis. 77 Osteoblasts differentiated from MSCs are the main sources of bone formation, and disruption to the MSC-osteoblast differentiation process leads to improper bone formation and imbalanced bone homeostasis. 78 Increasing evidence had indicated that UPS is essential for fine-tuned regulating of MSC differentiation, 79 with E3 ligases ubiquitinating self-renewal-associated regulatory proteins to promote stem cell differentiation, while DUBs stabilize proteins to promote cell self-renewal and stemness. 80 As the most extensively studied DUBs, several USPs, such as USP1, 15 USP7, 20 USP11, 27 USP15, 32 USP26, 13 USP 34 81 and USP53, 36 have been found to regulate the self-renewal and osteogenic differentiation of MSCs ( Figure 1).
SOX2 and NANOG are two proteins necessary for the self- T A B L E 2 Diseases associated with USPs mutation.

USP8
Cushing disease [55] USP9x Multiple epiphyseal dysplasia [56] USP9X-female syndrome [57] USP16 Down syndrome [58] USP18 Multiple sclerosis [59] Pseudo-TORCH syndrome [ Parkinson disease, late-onset [62] USP42 Acute myeloid leukaemia [67] USP44 Congenital heart disease [68] Intellectual disability [69,70] USP45 Leber congenital amaurosis [71] USP47 Amyotrophic lateral sclerosis [72] USP48 Crohn disease [73] USP53 Hereditary cholestasis [74,75] Progressive hearing loss [76] CYLD Crohn disease [50] Growth arrest is required for cells to undergo differentiation. 83 However, USP7 overexpression increased cell proliferation, which may be the reason that overexpressed USP7 did not enhance the differentiation of the BMSCs. 20 Another study, from Tang et al, showed that USP7 regulated not only the osteogenic differentiation of human BMSCs but also the osteogenic and adipogenic differentiation of adipose-derived stem cells. 23 However, the specific mechanism by which USP7 regulates the osteogenic differentiation of BMSCs is still unknown. Casein kinase 2 (CK2), a highly conserved serine/threonine kinase, is a negative regulator of BMP signalling and osteoblast differentiation. 84,85 CK2 phosphorylates RUNX2 to recruit the deubiquitinase USP7, which stabilizes the RUNX2 level by protecting it from ubiquitin-dependent proteasomal degradation. 22 Therefore, USP7 is likely involved in the osteogenic differentiation of MSCs via the deubiquitination and stabilization of RUNX2. 22 In addition, USP7 deubiquitinated and stabilized the expression of K-specific demethylase 6B (KDM6B) to promote the proliferation, differentiation and autophagy of osteoblasts. 21 15 However, USP1 levels have been shown to decline steadily during differentiation into F I G U R E 1 Effect of USPs on the self-renewal and osteogenic differentiation of MSCs. USP7 enhances the self-renewal capacity of BMSCs via the deubiquitination and stabilization of SOX2 and NANOG and is necessary in the early stages of the osteogenic, adipogenic and chondrogenic differentiation of BMSCs. USP7 also regulates BMP signalling pathway through deubiquitination and stabilization of KDM6B and RUNX2+, thus participating in osteogenic differentiation of BMSCs. In addition, USP34 interacts with Smad1 and RUNX2+, USP1 interacts with ID1/2, and USP11 interacts with MSX1 to inhibit their ubiquitination degradation and then regulate BMP signalling pathways. USP26 and USP53 regulate WNT/β-catenin signalling by inhibiting the ubiquitination degradation of β-catenin. USP18 and USP21 are also involved in osteogenic differentiation of MSCs. However, the specific mechanism remains unknown.
osteoblasts, which explains the reason that ID protein expression returns to basal level in the later differentiation stage. 15 Furthermore, the USP1 inhibitor ML323 enhances the osteogenic potential of human dental pulp stem cells. 89

| EFFECTS OF USPs ON OSTEOBLASTOGENESIS AND BONE FORMATION
In the skeletal system, osteoblasts are the main functional cells of bone formation, responsible for the synthesis, secretion and mineralization of bone matrix. 92 The proliferation, differentiation and maturation of osteoblasts are regulated by a variety of signalling pathways, such as PTH, WNT/β-catenin, BMP and Oestrogen, which are also regulated by USPs (Figure 2).
Parathyroid hormone (PTH) is an effective regulator of bone metabolism that effects osteoblast proliferation through the G protein-coupled transmembrane receptor PTH1R. 93 32 USP6, also known as TRE17, has been studied primarily in aneurysmal bone cysts. 104 USP6 potently inhibits the maturation of preosteoblasts via an autocrine mechanism involving BMP-4 dysregulation and the upregulation of the BMP antagonist Gremlin-1. 19 However, the mechanism by which USP6 regulated BMP action remains unclear.
Oestrogen also plays an important role in maintaining bone homeostasis. Recently, many investigators have shown an interest in the potential role of oestrogen in mediating cellular senescence, a key process in age-related diseases and bone loss. 26,105,106 The transcription factor p53 has been associated with cellular senescence and apoptosis and is a negative regulator of bone regeneration. 107 USP10, which can act as a senescence inducer, 108 promotes p21 transcription by directly removing ubiquitin molecules from p53 to maintain p53 stability and function. 109,110 Oestrogen increased the protein levels of p53 and p21 in MC3T3-E1 cells, mainly through a USP10-dependent deubiquitination pathway, to mediate p53 stability. 26 USP14 is also a potential DUB enzyme that acts on p53, and USP14 may contribute to dexamethasone-induced apoptosis in MC3T3-E1 cells by stabilizing p53 expression. 30

| EFFECTS OF USPs ON OSTEOCLASTOGENESIS AND BONE RESORPTION
Osteoclasts are multinucleated cells generated from monocyte/ macrophage precursor cells through a progressive differentiation process involving regulation by cytokines and transcription factors related to bone resorption and remodelling. 111,112 TRAF6 is a major adapter F I G U R E 2 Effect of USPs on osteoblastogenesis and bone formation. USPs regulates the proliferation, differentiation and maturation of osteoblasts via a variety of signalling pathways. USP2 protected PTHR from PTH-induced downregulation via direct deubiquitination of the receptor. USP4, USP7, USP8 and USP15 participate in the WNT/β-catenin by interacting with DVL, Axin, WNT and β-catenin and inhibiting their ubiquitination degradation, respectively. In addition, USP4 and USP15 can also participate in BMP signalling by interacting with Smad4 and ALK3 and inhibiting their ubiquitination degradation, respectively. USP10 and USP14 act as senescence inducers by stabilizing p53 expression and then promote the transcription of p21.
protein that mediates the signalling cascade after RANKL-RANK activation in osteoclasts. 113 TRAF6 binds to RANK to recruit and activate TGF-beta-activated kinase 1 (TAK1) and then activates inhibitor of nuclear factor kappa-B (IκB) kinase (IKK). 112 The IκB protein is subsequently phosphorylated, ubiquitinated and degraded to activate NF-κB. The active NF-κB protein is then transferred to the nucleus where it initiates the transcription of osteoclast-specific genes and ultimately induces the differentiation of osteoclast precursors into mature osteoclasts. 114 USPs also plays an important role in the proliferation, differentiation and maturation of osteoclasts ( Figure 3).
Our previous study showed that the expression of USP25 in peripheral blood monocytes is negatively correlated with bone mineral density (BMD) and positively correlated with TRAF6 expression in females, suggesting that USP25 may participate in the differentiation of monocytes into osteoclasts by stabilizing the expression of TRAF6. 14 SQSTM1/p62 (sequestosome 1) is a multifunctional ubiquitination-bound adaptor protein involved in the regulation of autophagy. 115 TRAF6 recruits SQSTM1/p62 to form a ternary complex with atypical protein kinase C (aPKC) and then activates NF-κB signalling. 116 The deubiquitination enzyme Cylindromatosis (CYLD) physically interacts with SQSTM1/p62 and is therefore recruited into TRAF6 to negatively regulate RANK signalling by antagonizing TRAF6-mediated polyubiquitination. 39 The TRAF6-p62-CYLD signalling complex is an important link in the NF-κB pathway of osteoclast differentiation and is currently an important target for developing new strategies for treatments of bone diseases. 117-122 B-cell Chronic lymphocytic leukaemia protein 3 (BCL3) is another protein that physically interacts with TRAF6 and is involved in non-canonical NF-κB signalling pathway. CYLD is assembled into TRAF6, which then removes the K63-linked polyubiquitin chain of BCL3, which is then transported out of the nucleus, resulting in downregulation of transcription of the BCl3-dependent cyclin D1 gene. 40 Other USPs, such as USP8 115 and USP36 123 were also found to deubiquitinate SQSTM1/p62 and suppress its autophagy activity, and USP20 was found to regulate the TNFα-Induced NF-κB Signalling Pathway by stabilizing the SQSTM1/ p62 protein. 124 However, their role in bone metabolism have not yet been extensively studied, and further study will deepen the understanding of the relationship between autophagy and osteoclasts.
USP18, also known as UBP43, was originally identified as a type I interferon-responsive gene. 125  bone destruction in mice with collagen-induced arthritis (CIA). 28 Proinflammatory NF-κB and PI3K/AKT signalling has also been shown to be among the most important contributors to induce particle-induced inflammatory reactions in macrophages and bone cell lysis. 133 Activation of the USP14-NLRC5 pathway inhibited proinflammatory cytokine expression, PI3K/AKT and NF-κB signalling activities, M1 polarization of macrophages and osteoclast formation, thereby inhibiting titanium particle-induced osteolysis. 29

| EFFECT OF USPs ON THE REGULATION OF OSTEOBLAST-OSTEOCLAST COUPLING
The mutual regulation between osteoblasts and osteoclasts is the basis of bone formation and bone resorption balance during bone remodelling. 139 Paracrine action of cytokines and secreted molecules is one of the main ways of osteoblast-osteoclast coupling. 140 RANKL, F I G U R E 4 Effect of USP4 secretion on sclerostin in osteocyte. Mechanical loading up-regulates the expression of PGE2, which binds to EP2 to inhibit TGFβ signalling through a CYLD-dependent proteasome mechanism, thereby inhibiting the transactivation of pSmad2/3 and subsequently inhibiting the expression of Sost. The phosphorylation of USP4 by Ck2 can also inhibit the transcription of Sost by inhibiting ubiquitination degradation of SIRT1. Sost then acts as an antagonist of WNT/β-catenin signalling and a promoter of RANK/RANKL signalling to modulate bone remodelling. In addition, USP53 is involved in the regulation of osteoblast-osteoclast coupling. Mechanistically, USP53 inhibits the interaction between Smad3 and VDR in osteoblasts, resulting in reduced binding of VDR to its cognate elements, thereby inhibiting the activation of RANKL.
which is secreted mainly by osteoblasts and osteocytes in bone, is a major regulator of osteoclastogenesis. A recent study by Hariri et al. 38 found that USP53 is involved in regulating osteoblast-dependent osteoclastogenesis by controlling the expression of RANKL. Mechanistically, USP53 inhibits the interaction between Smad3 and vitamin D receptors (VDR), resulting in reduced binding of VDR to its cognate elements, thereby inhibiting the activation of RANKL. 38 In addition, it also found that Smurf2 can regulate the ubiquitination of Smad3 and affect the expression of RANKL in osteoblasts by inhibiting the interaction between Smasd3 and VDR. 141  inhibits the expression of Sost by modifying the acetylation of H3K9 at the Sost promoter. 146,147 The phosphorylation of USP4 by casein kinase 2 (Ck2) inhibits the ubiquitination degradation of SIRT1, thus inhibiting the transcription of Sost and reducing the RANKL/OPG ratio in osteocytes, thereby reducing osteoclastogenesis and increasing osteoblastogenesis. 18 Overall, Sost expression regulated by CK2-USP4-SIRT1 pathway is essential for osteocytes to maintain bone homeostasis.
Mechanical loading is also an important factor for the maintenance of bone mass. Osteocytes are bone-embedded cells that sense mechanical stimulation and respond by regulating the activity of osteoblasts and osteoclasts. 148 Prostaglandin E2 (PGE2) is rapidly upregulated in osteocytes upon mechanical stimulation. PGE2 acts through its receptor (EP2) and inhibits TGFβ signalling through a proteasome mechanism dependent on deubiquitinase CYLD, thereby inhibiting pSmad2/3 and its transactivation of Serpine1 in osteocytes. 149 The downregulation of Smad3 phosphorylation and activity thereby inhibited the expression of Sost in osteocytes. 150 Thus, mechanical loading maintains bone homeostasis through the TGFβ pathway to regulate Sost expression ( Figure 4).

| CONCLUSION
Recent studies and evidence continue to reveal the involvement of USPs as DUBs in bone metabolic processes (

CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.