There are >8.9 million osteoporotic fractures occurring annually in the world (1). A total of >33% of women and 20% of men >50 years old will suffer an osteoporotic fracture at least once in their lifetime (2). With the increase in population aging, osteoporosis has become a chronic disease involving fractures, high disability and mortality and increased social and economic burden (3). Osteoporosis is a multifactorial disease characterized by bone loss and damage to the microstructure of the trabecular bone, leading to an increased risk of fracture and bone fragility (4,5). Therefore, identifying the pathogenesis of osteoporosis is of high importance.

Osteoporosis is closely linked to imbalances in bone homeostasis. The maintenance of bone homeostasis mainly depends on the balance between osteoclasts and osteoblasts; osteoclasts mediate bone resorption and osteoblasts mediate bone formation. In organisms, bone tissue constantly undergoes remodeling process through the synergistic action of bone marrow mesenchymal cells (BMSCs), osteoblasts, osteocytes and osteoclasts (6). BMSCs can multi-directionally differentiate into osteogenesis, adipogenesis and cartilage. BMSCs also promote calcium deposition and bone formation (7). Osteoblasts are derived from BMSCs and play a key role in the process of bone formation. They are mainly distributed on the surface of bone and synthesize bone matrix by secreting collagen and bone matrix proteins. Osteoblasts can synthesize and secrete the main components of bone, including collagen fibers, chondroitin sulfate, phosphoric acid and calcium. These secretions are important components of the bone matrix and play an important role in maintaining the structure and function of bone. Osteoclasts, originating from the hematopoietic monocyte-macrophage system, are a special type of terminally differentiated cells. They are involved in the process of bone remodeling through the uptake of bone matrix and minerals, as well as the secretion of organic acids and protein hydrolyzing enzymes, thus maintaining the normal structure and function of the skeleton (8). As osteoblast-derived cells, osteocytes maintain mature bone metabolism by mediating cytokine sensing, transduction and secretion to modulate bone resorption and formation (9). Under pathological conditions, osteoclast activity increases, while osteogenesis is either weakened or impaired, consequently destroying bone homeostasis. Over time, osteoporosis develops further.

During bone homeostasis and repair, Wnt/β-catenin, Hedgehog, bone morphogenetic protein (BMP-2)/Smad and phosphatidylinositol-3-kinase/protein kinase B and others are the major regulatory signaling pathways during skeletal growth and development. The Wnt/β-catenin signaling pathway plays a key regulatory role in bone development and formation by affecting the proliferation and differentiation of osteoblasts and osteoclast generation. Wnt is composed of 19 secreted glycoproteins and has the function of regulating cell growth, differentiation and apoptosis (10,11). The receptor activator of nuclear factor-κB ligand (RANK-L)/RANK/MAPK and the NF-κB axes are the major regulatory signaling pathways in osteoclast formation and function. RANKL integrates with the osteoclast surface receptor RANK, which in turn recruits tumor necrosis factor receptor-associated factor (TRAF) 6 and further activates multiple downstream signaling pathways, such as three mitogen-activated protein kinases (MAPKs) including p38 kinase (p38MAPK), extracellular signal-regulated kinase (ERK) and C-Jun N-terminal kinase (12,13).

Ubiquitination is a conserved, widespread and dynamic post-translational modification involved in regulating the activity and stability of thousands of proteins, thereby affecting most cellular functions and responses (14). Ubiquitylation is a process mediated by the covalent attachment of ubiquitin (Ub) to the target protein, catalyzed by a series of enzymatic cascades, including E1-mediating activation, E2-mediating coupling and E3-mediating connection step (Fig. 1) (15). E3 then targets the protein by promoting the formation of an isopeptide bond between the C-terminal carboxyl group of Ub and one of the seven lysine residues of Ub (K6/11/27/29/33/48/63) or the N-terminal methionine of Ub (M1) (16-18). Subsequently, modified substrate proteins are degraded by 26S protein complexes or exhibit cellular function (19,20). E3 provides the reaction rate and substrate specificity for this cascade reaction. Accumulating evidence has shown that E3 ligases are associated with a number of biological processes, including bone homeostasis (21). There are 600-1,000 E3 ligases encoded by the human genome. According to the domain structure and ubiquitination mechanism through which E3 ligases interact with target proteins, E3 members are broadly divided into three major subfamilies: i) Homology to E6AP C-Terminus (HECT) family; ii) really interesting new gene figure (RING) family; and iii) RING-between-RING (RBR; Fig. 2). The reversibility of ubiquitination modification is achieved by deubiquitinating enzymes (DUBs) (22). At present, >100 DUBs are encoded in the human genome based on the catalytic region. They can be classified into two categories or six subfamilies (Fig. 3) (23-28).

In recent years, a growing body of evidence suggests that ubiquitination plays an important regulatory role in the maintenance of bone homeostasis. Osteoporosis and other related bone diseases are accompanied by abnormal expression and dysfunction of ubiquitination and de-ubiquitination enzymes. As a potential research hotspot for bone metabolism-related diseases and a new target for drug development, ubiquitination has a broad prospect in the field of osteoporosis prevention and treatment. The current review discusses the specific mechanism and role of ubiquitination in bone remodeling to provide a more theoretical basis and practical guidance for the treatment of related diseases (Fig. 4; Table I).

In a growing number of studies, bone remodeling has been linked to ubiquitination. E3 Ub ligases are mainly involved in osteoblast differentiation and bone formation. In the present review, the function and regulation of E3 ligases in bone homeostasis is discussed.

DUBs are involved in DNA repair, gene transcription, apoptosis, autophagy, DNA repair and immune responses (19). DUBs act as critical regulators for bone remodeling by regulating basic multicellular unit differentiation and/or function.

The main goal of osteoporosis medications is to reduce the risk of osteoporotic fractures and improve the quality of life of patients by relieving pain, increasing bone density and preventing fractures (94). According to the different mechanisms of action, anti-osteoporosis drugs are generally placed in three categories: i) Inhibitors of bone resorption; ii) promoters of bone formation; and iii) dual-acting drugs. Bone formation promoters are mainly parathyroid hormone receptor agonists, including teriparatide and appalatide. Sclerostatin monoclonal is a double-acting drug (95). There are five main categories of bone resorption inhibitors: i) Bisphosphonates (BPs); ii) RANKL inhibitors; iii) estrogen; iv) selective estrogen receptor modulators; and iv) calcitonin (Table II).

BPs are widely used in the treatment of osteoporosis, where they are effective in inhibiting bone resorption and increasing bone mass at vertebral, non-vertebral and hip sites, thereby reducing the risk of fracture (96). However, oral BPs can increase upper gastrointestinal symptoms in patients and ~33% of patients receiving first intravenous zoledronic acid develop flu-like symptoms. In addition, prolonged use of BP medications may result in reduced blood supply to the jaws, thereby increasing the risk of osteonecrosis of the jaws as well as inhibition of bone turnover, leading to impaired bone remodeling, thereby increasing the risk of atypical fractures (97). Denosumab is also the most common osteoporosis drug in the clinic. It can be combined with RANKL to markedly reduce the degree of osteoporosis in patients. If Denosumab was stopped without other osteoporosis drugs, bone mineral density showed a steep decline (98). It is noteworthy that rashes, cellulitis, femoral shaft or subtrochanteric fractures and jaw necrosis may occur during use. The application of estrogen in women aged <60 years or 10 years after menopause can effectively prevent bone loss caused by menopause. Estrogen acts by indirectly regulating calcitonin, parathyroid hormone and 1,25-(OH)2D3. However, Estrogen replacement therapy has been limited clinically due to potential adverse effects, including venous thromboembolism, cerebrovascular disease, stroke and breast cancer (99). Menopausal hormone therapy (MHT) has its unique advantages in the treatment of postmenopausal osteoporosis (100,101). In the mid-1990s and early 2000s, in most European and North American countries, MHT is widely used to prevent the risk of postmenopausal osteoporosis and related bone fractures in women. Following the publication of the randomized Women's Health Initiative (WHI) report (102), which found that MHT increased the risk of coronary heart disease, stroke and breast cancer, there was a dramatic decline in MHT prescribing worldwide. The current trend is to recommend lower doses of estrogen for effective relief of vasomotor symptoms and prevent vaginal atrophy with an improved bleeding profile than with higher estrogen dosages within 10 years of menopause than was recommended a few years ago. For the prevention of endometrial cancer caused by MHT, it is recommended to add progesterone (103). In addition, compared with oral estrogen therapy, transdermal estrogen patch can reduce the risk of venous thrombosis and atherosclerotic vascular disease and also protect the heart (104). Therefore, low-dose non-oral MHT may be considered for women with cardiovascular risk factors, older women and those who choose to use MHT for extended periods to protect bone (105,106). In conclusion, individualized treatment is advocated for hormone replacement therapy and different formulations, dosages and MHT regimens are considered at the same time. The artificial synthesis of selective estrogen receptor modulators can selectively act on different tissue estrogen receptors to reduce the incidence of breast cancer in women (107). However, some side effects, including vasomotor symptoms, muscle spasm and venous thrombosis, remain. The Use of Raloxifene in Heart Disease (RUTH) study showed that Raloxifene increased fatal stroke (0.7‰ increase in absolute risk) and the risk of the occurrence of venous thromboembolic disease (absolute risk increase by 1.3‰) (108).

Parathyroid hormone is one of the important hormones that regulate calcium and phosphorus metabolism, as well as bone conversion. It has been shown that parathyroid hormone promotes bone anabolic metabolism that is dependent on low-dose and intermittent administration. The duration and dosage of teriparatide use are closely associated with rat bone tumors (109), but post-marketing surveillance did not reveal a higher than expected risk of osteosarcoma (110). Calcitonin reduces blood calcium and phosphorus mainly by inhibiting osteoclasts. However, long-term use of calcitonin leads to clinical resistance. In addition, there may be an increased risk of cardiovascular complications. This may be associated with the effect of calcitonin on the cardiovascular system; it may affect the systolic and diastolic function of the heart, or the relaxation and contraction of blood vessels (111). Monoclonal sclerostin romosozumab promotes the formation of bone mass, while it inhibits the resorption of bone, rapidly forming bone on trabecular and cortical bone surfaces to increase bone density and strength (112). The most common adverse reactions include arthralgia, nasopharyngitis, back pain and injection site reactions, rare mandibular osteonecrosis and atypical fractures. The black box warning on the potential cardiovascular risks of romosozumab, including serious adverse events such as non-fatal myocardial infarction, non-fatal stroke and cardiovascular death, may indeed place some limitations on its clinical use (95). Osteoporosis guidelines emphasize vitamin D and calcium supplementation as basic measures. Importantly, it has been shown that when patients with osteoporosis are treated with anti-osteoporosis drugs, the effectiveness of these drugs is markedly reduced without adequate vitamin D and calcium supplementation (113).

UPS is critical for bone cell formation and differentiation. Based on these mechanisms of action, drugs targeting these Ub ligases and deubiquitinating enzymes for the treatment of osteoporosis and other related bone diseases continue to be developed. For example, by inhibiting the activity of certain Ub ligases, it may be possible to promote bone formation or inhibit bone resorption for the treatment of osteoporosis. By contrast, by activating the activity of certain deubiquitinating enzymes, it may also be possible to have a positive impact on the treatment of skeletal diseases.

Catalpol, a biologically active cyclic enol ether terpene glucoside extracted from the traditional Chinese Medicine Rehmannia glutinosa, inhibits the ubiquitination and degradation of phosphatase and tensin homolog to increase phosphatase activity and then inhibits downstream AKT and NF-κB associated with RANKL signal pathways, consequently suppressing osteoclast formation and bone absorption (114). In mice with LPS and ovariectomized-induced bone loss, catalpol improves bone loss by attenuating osteoclast activity (114). Melatonin is a biologically active amine hormone secreted by the pineal gland of the brain and is a signaling molecule with a circadian rhythm, participating in various physiological processes including bone metabolism (109). Melatonin inhibits RANKL receptor activators to inhibit osteolysis and prevent bone loss and also promotes osteoblast differentiation and activity by melatonin receptor 2 (115). Melatonin reduces the ubiquitination of the Smad1 protein preventing its degradation by inhibiting Smurf1 activity, thereby stabilizing BMP Smad1 signaling activity and restoring inflammatory factor-induced osteogenesis (116). Carnosic acid (CA), an amino acid substance with strong antioxidant and anti-aging effects, is widely used in weight loss foods, nutritional supplements and other fields. CA binds to the ligand-binding region of cholesterol and estrogen receptor-associated receptor α, promoting its ubiquitination and proteasomal degradation, thereby inhibiting RANKL-associated osteoclast formation and ovariectomy-induced bone loss (117).

Beraprost can improve postmenopausal osteoporosis by downregulating the NEDD4-mediated ubiquitination-proteasome degradation of Runx2 (118). The selective complexes B06 and B75 block binding of Smurf1 to Ub, thereby stabilizing the level of the BMP signaling component Smad1/5 protein and enhancing osteoblast activity. This also offers promising new clinical prospects for the therapy of osteoporosis (119). miR-195-5p can target and inhibit the activation of BMP-2/SMAD/Akt/Runx2 pathway signaling by Smurf1, to promote osteogenic differentiation in ovariectomized mouse models (120). Chalcone derivatives enhance local bone mass after lumbar spine fusion surgery in normal BMP-2 mice with high expression of Smurf1 (31). Bortezomib, is a 26S proteasome inhibitor which inhibits the degradation of polyUb protein by suppressing the hydrolysis activity of the proteasome. Bortezomib regulates proteasomal degradation of Runx2 via the Wnt/β-catenin signaling pathway (121), inducing osteogenic differentiation and promoting osteogenesis (122). Furthermore, a recent study suggested that bortezomib may treat post-menopausal osteoporosis by inhibiting the Smurf-mediated ubiquitination-proteasome process (123). Withaferin A, also a proteasome inhibitor, promotes osteoblast proliferation and differentiation as well as regulates bone anabolism to promote osteoporotic fracture healing (124).

miRNAs participate in the progress in osteoporosis through a complex signaling pathway network (125). miR-21-5p inhibits osteoclast differentiation by targeting SKP2. In an ovariectomy (OVX) mouse model injected with miR-21-5p, the number of osteoclasts was reduced and the degree of osteoporosis was improved (126). Neat1, the mechanoreceptor long non-coding RNA (lncRNA), under mechanical stimulation, regulates osteogenic function by nuclear retention of the paraplaque-dependent E3 Ub ligase Smurf1 mRNA. In Neat1-knockout hindlimb unloading mice, no bone damage or bone loss are found. This provides a treatment reference for bone loss caused by long-term space flight or inactivity and osteoporosis associated with aging (127). In addition, SNHG1 lncRNA inhibits osteogenic differentiation through NEDD4-mediated ubiquitination by suppressing the activation of the p38-MAPK signaling pathway, a key trigger factor in bone formation (128). In recent years, a new drug delivery system, nanoparticles, has been used to treat osteoporosis. Ferumoxytol and ferucarbotran, as clinical nanoparticles, exert anti-osteoporosis effects by inhibiting osteoclastogenesis and participating in bone metabolism. Nanoparticles trigger the upregulation of p62, resulting in the recruitment of CYLD to enhance deubiquitination and inactivation of TRAF6, the main controller of the RANKL and NF-κB signal (129). When downstream activation of the NF-κB signaling pathway and the MAPK signaling pathway is attenuated, the proliferation and differentiation processes of osteoclast precursors may be impeded, leading to a reduction in osteoclast formation. Intravenous injection can improve bone quality in OVX mice and it is anticipated to be an alternative drug for the treatment of osteoporosis (129).

In addition, several clinically used drugs have been discovered to be involved in bone homeostasis via the UPS system, such as thalidomide (130), lansoprazole (131), vitisin A, clomimidazole and zoledronic acid (132), all of which inhibit or induce ubiquitination of related substrates.

The present review introduced and summarized the current literature on ubiquitination and deubiquitination in osteoporosis and bone homeostasis, highlighting the critical importance of Ub-dependent proteolytic systems in skeletal cell function. The E3 connecting enzymes are mainly regulated by transcription factors and they have notable protein participation in bone formation in the signaling pathway of BMSC and osteoblast proliferation and differentiation. Only a few studies have shown that RNF146 (44) and Smurf2 (35) regulate osteoclasts. The E3 protease system can influence the bone remodeling process by regulating different target proteins. They recognize and degrade proteins that are involved in bone formation, thereby regulating the rate and extent of bone formation. At the same time, they also recognize and degrade proteins involved in bone resorption, thereby controlling the process of bone resorption. This precise regulatory mechanism is essential for maintaining bone homeostasis.

Research on the regulation of deubiquitinating enzymes on osteoblasts and osteoclasts is limited and the mechanism and molecules of action have not been fully elucidated. To date, the relationship between MJDs and MINDY families with skeletal cell differentiation and function has not been reported. Elucidating the intrinsic mechanisms by which DUB gene alterations regulate osteoblast and osteoclast functions is important for the development of novel and more effective therapeutic strategies for osteoporosis. Designing drugs targeting specific genetic alterations in the DUB gene to avoid its dysregulation is one of the important directions for developing novel osteoporosis treatment strategies. Through in-depth study of the function and regulatory mechanisms of the DUB gene, potential drug targets can be identified and targeted drugs can be designed to regulate the function of osteoblasts and osteoclasts. These drugs can correct the imbalance of bone metabolism by altering the activity or expression level of the DUB gene, thus achieving the goal of treating bone diseases such as osteoporosis. In the future, through in-depth study of the function and regulatory mechanism of the DUB gene, as well as the design of drugs targeting its specific genetic alterations, it is anticipated to provide new ideas and methods for the treatment of skeletal diseases such as osteoporosis.

Meanwhile, several anti-osteoporosis drugs commonly used in the clinic were discussed in the present review as well as their disadvantages and the current clinical studies on ubiquitination-related drugs for osteoporosis. At present, anti-osteoporosis drugs have shown some efficacy in clinical research and their benefits outweigh the risk of side effects, but patients lack compliance due to the aforementioned side effects, high costs and sequential treatment. Although designing drugs to target specific genetic alterations in the DUB gene is challenging and the adverse effects and the interactions with other drugs are not yet clear, the future research direction may be to treat osteoporosis by mediating ubiquitination.

Furthermore, the internal mechanisms by which ubiquitination regulates skeletal cell function have not been thoroughly investigated and the specific sites of ubiquitination modification and the interactions of different molecules acting at the same site have not been clarified. Decoding the pathways between ubiquitination and other modification mechanisms has also not been described in depth. These practical mechanisms should be further investigated in the future.