The role of retinoic acid receptor-related orphan receptors in skeletal diseases

Bone homeostasis, depending on the balance between bone formation and bone resorption, is responsible for maintaining the proper structure and function of the skeletal system. As an important group of transcription factors, retinoic acid receptor-related orphan receptors (RORs) have been reported to play important roles in bone homeostasis by regulating the transcription of target genes in skeletal cells. On the other hand, the dysregulation of RORs often leads to various skeletal diseases such as osteoporosis, rheumatoid arthritis (RA), and osteoarthritis (OA). Herein, we summarized the roles and mechanisms of RORs in skeletal diseases, aiming to provide evidence for potential therapeutic strategies.


Introduction
Bone homeostasis is an essential dynamic process of bone modeling and remodeling (1), which requires accurate regulation by a variety of transcription factors as well as signaling pathways (2,3).Retinoic acid receptor-related orphan receptors (RORs) are ligand-dependent transcription factors and can regulate osteogenesis and osteoclastogenesis in bone metabolic disorders such as osteoporosis (4).In addition, the RORs, particularly RORg, mediate the inflammatory response and lead to bone destruction in inflammatory bone diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA) (5).In this review, we discussed the effect of RORs on maintaining bone homeostasis and summarized prospective therapeutic strategies targeting RORs for common bone diseases.
The modular structure of RORs is typical of NRs and consists of four functional domains: a distinctive N-terminal domain, a highly conserved DNA-binding domain (DBD), a ligand-binding domain (LBD), and a hinge domain (Figure 1A).Differences in sequence of the N-terminal domain determine the isoforms of RORs.ROR response elements (ROREs) have an AGGTCA consensus motif with an A/T-rich region.With two highly conserved zinc finger motifs, The DBD is responsible for recognizing the AGGTCA consensus region in ROREs as a monomer.The LBD is the most important functional domain that is crucial for ligand-binding and facilitates the recruitment of cofactors in transcriptional regulation.The hinge domain linking DBD with LBD stabilizes the RORs' protein structure (8).
Even though evidence has indicated that RORs can be selfactivated without any ligands or other potential stimulators, RORs transcriptional regulation is primarily ligand-dependent (6,7).Cholesterol and its metabolites were firstly identified as natural ligands by crystallography studies (8).Melatonin was once considered as an endogenous ligand of RORb.Although it could bind to RORb directly, the result was not reproducible and therefore has been withdrawn (9).Apart from natural ligands, synthetic ligands have made significant progress (10).According to conformational changes of RORs, the ligands include agonists, antagonists, and inverse agonists (11).
RORs interact with co-repressors as well as co-activators and function as repressors or activators of gene transcription respectively.When inverse agonists enter the nucleus, RORs can be trans-activated and recruit co-repressors, such as the nuclear receptor co-repressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) (4).The corepressor complexes then combine with the RORE of target genes that subsequently prevent the promoter initiation and suppress the transcription.In contrast, agonists activate RORs with conformational changes resulting in the dissociation of corepressor complexes and the recruitment of co-activators, such as the p160 steroid receptor co-activator (SRC) family, p300, and BRG2 complex (6,12).The formation of co-activator complexes eventually facilitates transcriptional regulation by enhancing the promoter of target genes (Figure 1B).
The RORs serve functions in several physiological processes such as cell division and differentiation, circadian rhythm, metabolism, and immune regulation (12).ROR family members and their isoforms exhibit a distinct tissue-specific pattern of expression and play different roles in pathological processes.Specifically, RORa is widely distributed in multiple tissues, including the liver, skeletal muscle, skin, and adipose, which is  mainly associated with circadian rhythm abnormalities, tumorigenesis, and metabolic diseases.In addition, RORa is highly expressed in the pre-hypertrophic and hypertrophic chondrocytes of the growth plate, suggesting that RORa may be a regulator of hypertrophic differentiation of chondrocytes and contribute to endochondral ossification (13,14).RORb is expressed restrictedly in the brain, retina, bone, and pineal gland, and is essential for retinal cell survival and circadian rhythm regulation (4).Moreover, the two isoforms of RORg differ in the expression with distinct N-terminus.RORg1 is expressed in various tissues such as skeletal muscle tissue, liver, and kidney, while RORg2 (same as RORgt) is exclusively expressed in immune cells and plays prominent roles in thymocyte development (12).

Role of RORs in skeletal diseases
Skeletal diseases, such as osteoporosis, rheumatoid arthritis, and osteoarthritis, significantly impair individuals' health and quality of life.Patients may suffer from long-term pain, limitation of movement, and hypofunction.Studies have found that RORs play an important role in bone metabolism and maintenance of bone homeostasis.Interestingly, ROR family members play different roles in regulating osteogenesis.Most studies believe that RORa mainly promotes osteogenesis.On the contrary, RORb inhibits osteogenic differentiation.RORg regulates bone balance by participating in the inflammatory response and stimulating osteoclastic differentiation.Herein, we summarize the role of RORs in common skeletal diseases.

Osteoporosis
Osteoporosis is one of the most common bone metabolic disorders, characterized by bone loss and microstructural changes, which in turn increase the risk of fractures (15,16).Osteoporosis occurs when the rate of bone resorption consistently exceeds the rate of bone formation.RORs affect the occurrence and progression of osteoporosis by regulating the proliferation and differentiation of skeletal cells and multiple osteogenic signaling pathways.

RORa in osteoporosis
Studies have shown that RORa positively regulates osteogenic differentiation in vitro and in vivo.Spontaneous mutations of the Rora gene were identified in a staggered (sg) mouse strain.Homozygous staggerer (sg/sg) mice showed limited skeletal development with a reduction in long bone thickness and bone mineral density (17,18).Application of cholesterol sulfate, a RORa agonist, prevented bone mass loss in the inflammation-mediated or ovariectomy (OVX) -mediated osteoporosis models (19).Studies in goats have also confirmed a substantial association between RORa and growth traits including height and bone length (20).In vitro evidence suggested that RORa was highly expressed in human mesenchymal stem cells (hMSCs) and that the expression level increased with osteogenic differentiation (17,21).
Several upstream signaling pathways are mediated by RORa in osteogenic differentiation (Figure 2A).(1) Circadian clock system: Brain and muscle ARNT-like protein 1 (Bmal1) knockout mice exhibited osteoporosis (22).Further studies found that BMAL1 blocked the Wnt/b-catenin signaling pathway by inhibiting the expression of RORa, thereby preventing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) (23).( 2) Oxidative stress: It has been reported that oxidative stress is a key trigger of osteoporosis.OSGIN2, an inducer of oxidative stress, prevented osteogenic differentiation of BMSCs by inhibiting RORa expression (24,25).( 3) DNA methylation: Histone methylation has been recognized as an important modulator in the osteogenic differentiation of MSCs (26).RORa mediated DNA methyltransferase 1 (DNMT1) to control the DNA methylation of MSCs, thereby suppressing chondrogenic and osteogenic differentiation (27).( 4) Estrogen: Estrogen plays a significant role in regulating bone metabolism (28).Estradiol is one of the Estrogen's natural products, which could activate RORa and promote osteoblast differentiation (29).
There are several main mechanisms underlying the downstream pathway of RORa.One is directly promoting the transcription of osteogenic differentiation marker genes, such as alkaline phosphatase (ALP), collagen type I (COL I), bone sialoprotein (Bsp), and dentin matrix protein 1 (DMP1) (17,21,30).The second is activating osteogenic differentiation signaling pathways, such as Wnt/b-catenin and BMP2/Smad1/5/9 pathways (17, 29).Furthermore, RORa can associate with a variety of cell types and influence the microenvironment directly or indirectly.For instance, in the diabetic bone defect model, underexpression of RORa in macrophages inhibited the transcription of serum C-C motif chemokine 3 (Ccl3) and interleukin-6 (Il-6), which led to the inability of macrophages to convey migration and aggregation signals to BMSCs, ultimately leading to diabetic bone aplasia (31).
However, it has also been reported that RORa negatively regulates osteogenic differentiation.For example, transient overexpression of RORa suppressed osteocalcin (Ocn) transcription induced by vitamin D3 and consequently inhibited osteogenesis (17).In addition, bisphenol A (BPA), a ROR agonist, could promote bone resorption (32).The controversial results may be due to the time and extent of RORa expression in two different ways of both bone formation and resorption in bone metabolism.

RORb in osteoporosis
In contrast to RORa, RORb negatively regulates osteogenic differentiation and promotes the progression of postmenopausal or age-related osteoporosis.Deletion of the Rorb gene could prevent bone loss in aged mice (33).RORb expression was significantly increased with age in the BMSCs from postmenopausal women, which was consistent with the mouse models of age-related osteoporosis (34,35).Data on overexpression or knockdown of RORb in osteoblastic MC3T3-E1 cells also suggested its negative effect on osteogenic differentiation (33,36).
The following mechanisms may explain the negative effects of RORb on osteogenic differentiation (Figure 2B).(1) Runt-related transcription factor 2 (Runx2) is an essential transcription factor that drives the phenotypic expression of osteoblasts.Its absence can lead to a complete loss of ossifying capacity (37).When the expression of RORb was up-regulated in MC3T3-E1 cells, transcription of Runx2 and its target genes Ocn and osterix (Osx) were significantly inhibited (34).( 2) Transforming growth factor b (TGF-b) and bone morphogenetic protein (BMP) control the formation of extracellular matrix (ECM) and provide a framework for bone mineral deposition (38).Studies have shown that overexpression of RORb upregulated the expression of these cytokines and then interfered with ECM synthesis, thereby inhibiting bone mineralization (36).(3) RORb down-regulated the Wnt/b-catenin signaling pathway by inhibiting the activities of Wnt downstream target genes transcription factor 7 (Tcf7) and osteoprotegerin (Opg), which disrupted the balance between bone formation and bone resorption, leading to bone loss (33).( 4) RORb could stimulate osteoblast proliferation through the mitogenactivated protein kinase (MAPK) pathway and prevent osteoblasts from exiting the cell cycle, thereby delaying osteogenic differentiation (36).In addition, RORb has been identified as a molecular target of miR-219a-5p in recent years, which is involved in the development of osteoporosis (39).

RORg in osteoporosis
Most studies on the effect of RORg on osteoporosis were limited to RORg2 (RORgt).The main mechanism is to act as a specific transcription factor of immune cells, especially T helper cell 17 (Th17), and mediate the differentiation of Th17 cells to secret interleukin 17 (IL-17), thereby enhancing inflammatory response (5).In recent years, it has been found that T cell-mediated inflammation plays an important role in the progression of osteoporosis (40, 41).In human and mouse models, RORgt affected bone loss in postmenopausal osteoporosis caused by estrogen deficiency (42,43).The production of IL-17, mediated by RORgt, directly stimulated osteoclastogenesis by upregulating the expression of RANKL (44,45).IL-27 inhibited the differentiation of Th17 cells in OVX mice by inhibiting the transcription factor RORgt (46).In addition, as one of the areas of scientific interest in recent years, intestinal flora plays a key role in regulating bone homeostasis through the gut-bone axis (47).The treatment of Lactobacillus rhamnosus GG (LGG) stimulated Th17 cell differentiation with the alteration of RORgt expression, and had an advantage in osteogenic promotion, which may account for the amelioration of osteoporosis in OVX model rats (48,49).Another popular probiotic, Bifidobacterium longum (BL), appeared its immunomodulatory potential to the Th17 cell differentiation that suppressed osteoclastogenesis in postmenopausal osteoporosis (50).

Rheumatoid arthritis
Rheumatoid arthritis is a systemic autoimmune disease characterized by initial synovial inflammation followed by cartilage degeneration and subchondral bone destruction (51,52).The promotion of osteoclast formation and differentiation is the main reason for bone erosion (53).Immune cells, especially Th17 cells, are activated and concentrated in the inflamed synovium, contributing to persistent joint inflammation and bone erosion in RA (54).Among RORs, many indicated the roles of RORa and RORgt in inflammatory diseases like RA and OA, whereas little has been done on RORb.

RORa in rheumatoid arthritis
RORa is involved in the inflammatory response stage of RA and the regulation of osteoclast activity in bone destruction.RORa is expressed in Th17 cells and the expression is elevated in collageninduced arthritis (CIA) mice (55).RORa can act as a transcription factor to mediate the differentiation of Th17 cells.The expression of RORa and RORgt together promoted the differentiation of Th17 and significantly up-regulated the expression of IL-17, thereby promoting inflammatory response (56).However, some studies have also reported that RORa is a negative regulator of inflammatory response.Compared with controls, the expression level of RORa in osteoblasts of RA patients was lower (30).The RORa agonist, cholesterol sulfate, could reduce joint inflammation and bone destruction in CIA mice by inhibiting RORgt expression and Th17 cell differentiation (57).Differences in the disease itself and the immune microenvironment determine the cellular response to any stimulus, which may help to partially explain the different mechanisms by which RORa regulates inflammation in Th17 cell differentiation.In addition, RORa is involved in the process of bone resorption in RA.RORa agonists prevented osteoclastogenesis and induced osteoclast apoptosis in CIA mice (57).However, direct depletion of Rora did not affect the agonist-ligand-mediated inhibition of osteoclast differentiation (19).This suggested that RORa agonists might be involved in RA osteoclast survival and differentiation in a RORa-independent manner.

RORg in rheumatoid arthritis
RORgt, as a lineage-specific transcription factor of Th17 cells, regulates the differentiation of CD4+ T cells into Th17 cells, then promotes the production of IL-17, and finally aggravates joint inflammation and bone destruction in RA (58) (Figure 2C).In RA patients, the aggravation of inflammation is closely related to the increase in the proportion of Th17 cells and the expression levels of RORgt and IL-17 (5,59).
In RA, upstream cytokines stimulated signal transduction and activation transcription factor 3 (STAT3) into the nucleus and initiated RORgt activation (61, 65).RORgt and STAT3 could bind in the IL-17 promoter region and thus regulate the production of IL-17.In the downstream pathways, IL-17 overexpression promoted the survival and maturation of fibroblasts and immune cells in the synovium, thereby aggravating synovial inflammation [90], which ran through the whole process of RA (76).At the same time, increased IL-17 levels might promote osteoclast generation through the RANKL/OPG signaling pathway, thereby aggravating subchondral bone erosion (77).Metallothionein-1 (MT-1) inhibited Th17 differentiation by reducing the expression of STAT3 and RORgt, and significantly suppressed synovial inflammatory response and subsequent bone destruction (78).

Osteoarthritis
OA is a chronic degenerative arthritis that affects all joints and is characterized by cartilage degradation, subchondral bone remodeling, and bone mineralization (79).Different from RA, where synovial inflammation is the main pathological change, cartilage degeneration is the pathological center of OA (80).The molecular mechanisms underlying OA pathophysiology are poorly understood.OA was previously considered to be a noninflammatory joint disease.However, recent studies have identified proinflammatory cytokines and immune inflammatory cells as key mediators of cartilage damage in OA (81).OA and RA share some risk factors and pathogenic features, and they both exhibit highly inflammatory features driven by CD4+ T cells (82).The expression of IL-17 in the synovial tissue of OA and RA patients was increased.This eventually led to increased joint inflammation and bone erosion (83).
It has also been suggested that metabolic disorders may also be an important mechanism leading to this disease.RORa can significantly affect the progression of OA through cartilagespecific cholesterol metabolism.In the CH25H-CYP7B1-RORa axis of cholesterol metabolism, RORa acted as a substrate for cholesterol hydroxylase (CH25H and CYP7B1) (84).High cholesterol and its metabolites could directly activate RORa in chondrocytes, which could bind to the promoter of cartilage matrix catabolic factors and activate its transcription, leading to cartilage damage (85,86).In addition, RORa accelerated cartilage matrix degradation through the IL-6/STAT3 signaling pathway (87, 88).

Therapy strategies targeting RORs in skeletal diseases
Therapeutic applications targeting RORs have developed along with the identification of ROR ligands including agonists, inverse agonists, and antagonists.Specifically, ROR agonists enhance the levels of target genes by either promoting the recruitment of coactivator complexes or preventing the recruitment of corepressor complexes, whereas ROR inverse agonists do the opposite.ROR antagonists reduce the transcription by preventing the recruitment of any complexes (11,89).
Since the first ROR agonist was identified, RORs have been investigated as a potential therapeutic target for various diseases (90,91).Among bone diseases, inflammatory arthropathy has received the most attention.Treatment with RORa agonist cholesterol sulfate prevented osteoclast osteogenesis in CIA mice and protected against bone loss in postmenopausal osteoporosis (19).A dual inverse agonist of RORb and RORgt, N-(5-(arylcarbonyl)thiazol-2-yl)amides, has been identified to exhibit therapeutic potential in CIA mice (92).Compared with RORa and RORb, the treatment targeting RORgt is more commonly used in inflammatory and autoimmune arthropathies.
Several natural compounds have been identified as RORgtspecific modulators that can be used in the treatment of autoimmune diseases.Digoxin was the first RORgt inhibitor reported to reduce inflammation in CIA mice (66), however, its disadvantages were obvious cytotoxicity and limited therapeutic index.In addition, ursolic acid had anti-RORgt activity, but it could also activate glucocorticoid receptors as a side effect (93).To solve these problems, the synthesis of selective RORgt small molecule modulators has become a promising treatment for autoimmune diseases such as RA (94).In vivo data showed that amide drugs had good clinical efficacy as effective RORgt inhibitors (95).Phenylenediamine derivative of RORgt inhibitor reduced the severity of arthritis symptoms in mice (96).Moreover, imidazolopyridine analogs, a RORgt selective inverse agonist, alleviated the pathological symptoms of adjuvant arthritis (AIA) in rats (97,98).Recently, three azacyclic inverse RORgt agonists were identified by structure-activity relationship studies, which showed biologically similar efficacy in preclinical models of RA (99).

Discussion and conclusion
In summary, as transcription factors, RORs directly regulate osteogenic differentiation and osteoclastogenesis and associate with other factors or signals to indirectly mediate the destruction of cartilage and bone by immune-inflammatory responses.In terms of selective molecular modulators, RORg has made remarkable progress in autoimmune diseases.However, on the pharmacological front, there are concerns about the safety and efficacy of existing small-molecule drugs targeting RORg.In addition, the effects of administration form and ROR binding mode on clinical efficacy remain to be further studied.

1
FIGURE 1 Schematic diagram of structure and ligand-dependent mechanism of RORs.(A) Structure: Four functional domains constitute RORs, including a distinct Nterminal domain, a highly conserved DNA-binding domain (DBD), a ligand-binding domain (LBD), and a hinge domain.The numbers at the tail represent the total number of encoded amino acids in the corresponding ROR isoforms.(B) Mechanism: Inverse agonists trans-activate RORs to recruit co-repressors and inhibit transcription of target genes (left).Agonists activate RORs to recruit co-activators and initiate transcription of target genes (right).

2
FIGURE 2 Regulation of osteogenic differentiation by RORa and RORb and of Th17 cell differentiation by RORgt.(A) Upstream effectors including BMAL1, OSGIN2 and DNMT1 could suppressed osteogenic differentiation by inhibiting RORa, while Estradiol activated RORa to promote osteogenic differentiation.(B) RORb overexpression inhibited osteogenic differentiation by inhibiting Runx2 transcription, disrupting ECM synthesis, inhibiting Wnt/b-catenin pathway, and promoting proliferation through the MAPK pathway.(C) RORgt regulates the process of differentiation from CD4+ T cells to Th17 cells, and the consequent IL-17 secretion.The latter can influence the survival and maturation of immune cells and osteoclastogenesis, which thus aggravates joint synovial inflammation and bone damage in RA patients.BMAL1, Brain and muscle ARNT-like protein 1; DNMT1, DNA methyltransferase 1; Runx2, Runt-related transcription factor 2; ECM, Extracellular matrix; MAPK, Mitogen-activated protein kinase; Th17, T helper 17; IL-17, Interleukin 17; miRNAs, MicroRNAs.