Therapeutic potentials and modulatory mechanisms of fatty acids in bone

Abstract Bone metabolism is a lifelong process that includes bone formation and resorption. Osteoblasts and osteoclasts are the predominant cell types associated with bone metabolism, which is facilitated by other cells such as bone marrow mesenchymal stem cells (BMMSCs), osteocytes and chondrocytes. As an important component in our daily diet, fatty acids are mainly categorized as long‐chain fatty acids including polyunsaturated fatty acids (LCPUFAs), monounsaturated fatty acids (LCMUFAs), saturated fatty acids (LCSFAs), medium‐/short‐chain fatty acids (MCFAs/SCFAs) as well as their metabolites. Fatty acids are closely associated with bone metabolism and associated bone disorders. In this review, we summarized the important roles and potential therapeutic implications of fatty acids in multiple bone disorders, reviewed the diverse range of critical effects displayed by fatty acids on bone metabolism, and elucidated their modulatory roles and mechanisms on specific bone cell types. The evidence supporting close implications of fatty acids in bone metabolism and disorders suggests fatty acids as potential therapeutic and nutritional agents for the treatment and prevention of metabolic bone diseases.

blood. 1,2 Bone formation is initiated by bone marrow mesenchymal stem cells (BMMSCs) migrating from vascular channels circulation to bone surface. Osteoblasts deposit organic bone matrix and regulate its mineralization and eventually differentiate into osteocytes that are embedded in the cavities of mineralized matrix. 2,3 In addition to osteoclasts, BMMSCs and osteoblasts, other bone cell types participating in bone metabolism include macrophages, surface bone-lining cells, chondrocytes as well as osteocytes (Figure 1). [4][5][6][7][8][9][10] Accumulating evidence has established essential roles of fatty acids in bone metabolism 11 (Table 1). Categorization of fatty acids involved in bone metabolism has been reviewed by Natalia S.
Harasymowicz et al 12 In general, ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) are a group of well-known fatty acids obtained from diet and supplemented via in vivo synthesis, 13 and eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA) are the three major representatives of ω-3 LCPUFAs.
ω-3 LCPUFAs could mediate bone metabolism via processes including lipid oxidation, calcium absorption and prostaglandin synthesis, 14 and they can exert beneficial effects on bone remodelling by inhibiting osteoclast activity and enhancing osteoblast activity. 15 Several studies have investigated the therapeutic properties of ω-3 LCPUFAs. By promoting bone formation, ω-3 LCPUFAs significantly affect peak bone mass, 16 increase bone calcium levels as well as bone mineral content (BMC) and density. [17][18][19][20] Therefore, they represent a non-pharmacological strategy for preventing bone loss and accelerating fracture healing 21 and thus to reduce risks of osteoporosis and rheumatoid arthritis. 16,22,23 In addition, ingestion of ω-3 LCPUFAs eliminates adriamycin-or cyclophosphamide-induced toxicity in bone marrow and bone tissues, suggesting potential roles of ω-3 LCPUFAs in combating side effects of specific bone-targeted drugs. 24 Long-chain monounsaturated fatty acids (LCMUFAs) such as ω-5, ω-7 and ω-9 categories are commonly recognized as potential agents against osteoporosis and other osteolytic diseases. They promote bone formation and inhibit bone degeneration and thereby facilitate bone metabolism. By contrast, long-chain saturated fatty acids (LCSFAs) might negatively affect bone metabolism. Intake of common dietary SFAs such as lauric acid (LA, C12:0), myristic acid (MA, C14:0), palmitic acid (PA, C16:0) or stearic acid (SA, C18:0) might initiate inflammatory osteoarthritis and obesity. [25][26][27] Moreover, medium-chain fatty acids (MCFAs) such as capric acid (CA) have been reported to suppress osteoclastogenesis and thereby alleviate bone resorption. Short-chain fatty acids (SCFAs) [28][29][30] including acetate, butyrate and propionate have been suggested to inhibit bone resorption and combat inflammation. As a result, SCFAs are promising in the prevention of inflammatory bone loss and arthritis. Furthermore, fatty acid derivatives such as lipoxin A 4 (LXA 4 ) and resolvin E1 (RvE1) have also been involved in bone resorption attenuation. Therefore, considering large quantities of fatty acids in our daily diets, it is worthwhile to understand influences of fatty acids on bone metabolism and the underlying mechanisms, for further exploring their beneficial therapeutic applications in a wide variety of metabolic bone disorders.

| Periodontitis
Periodontitis is a chronic bacterial infection disease characterized by primary gingival and extended alveolar bone inflammation, accompanied by periodontal tissue damage 31,32 with connective tissue degradation and even tooth loss. 33    LCPUFAs might not be applied to periodontitis prevention and treatment. 44,46 In mechanism, fatty acids might exert effects on periodontitis pathogenesis and intervention via direct and indirect mechanisms.
Fatty acids could directly affect periodontitis-associated bone destruction. LCSFAs such as PA could trigger P gingivalis-induced alveolar bone loss directly. 31 In benefit, EPA metabolite RvE1 could target BLT1 receptors in osteoclasts to inhibit osteoclast fusion and maturation, and RvE1 can induce the release of osteoprotegerin (OPG) to antagonize the proresorptive role of osteoclast-stimulating receptor activator of nuclear kappa-β ligand (RANKL), and thus facilitates the prevention of alveolar bone loss and enhances periodontal bone regeneration in periodontitis patients. 47 The indirect effects of fatty acids in periodontitis are mainly through inflammatory response.
Studies have shown that LCSFAs (such as PA) at high levels in plasma may facilitate P gingivalis-induced chemokine production in human gingival fibroblasts and further promote inflammatory response in periodontium. 31 PA-induced chemokine secretion in human gingival fibroblasts could be inhibited by LCPUFAs (such as DHA), and such effects presumably involving the suppression of toll-like receptor (TLR) dimerization as well as nuclear factor-kappa B (NF-κB) activation. 48 In addition to exert effects on chemokine, fatty acids such as RvE1 could also act on inflammatory cells by enhancing the migration of monocytes and neutrophils and promoting the clearance of apoptotic neutrophils to enhance pro-inflammatory response. 31

| Osteoporosis
Osteoporosis, marked by low bone mineral density (BMD) and deteriorated bone tissue microarchitecture, contributes to a high incidence of bone fracture on average up to 50% of women > 50 years. 50 Osteoporosis is mainly caused by excessive bone resorption resulting from imbalance between overactive osteoclasts and inactive osteoblasts. 51,52 Hence, inhibiting bone resorption or promoting bone formation are promising strategies for osteoporosis prevention and treatment. 53 It has been well acknowledged that osteoporosis is associated with levels of fatty acid in bone microenvironment. 54 As reviewed earlier by Salari et al, 55 investigations conducted in humans have shown inconsistent correlations between fatty acids and osteoporosis, while studies in animal models have confirmed that supplementation of ω-3 LCPUFAs alleviates osteoporosis by suppressing bone breakdown, promoting calcium absorption from diet, reducing prostaglandin E2 (PGE2) production and increasing skeletal calcium. 56 In mechanism, ω-6 LCPUFAs intake results in a high ratio of Estrogen deficiency-induced postmenopausal osteoporosis is the most common type of osteoporosis. Along with decrease in estrogen levels, reduction in OPG delays osteoblast maturation and attenuates bone formation 61 ; also, drop in OPG/RANKL ratio enhances osteoclast differentiation and promotes bone resorption and eventually results in bone loss. 47

| Rheumatoid arthritis
Rheumatoid arthritis, with manifestations of arthralgia, redness and swelling, and limited range of motion, 71 is a chronic and autoimmune inflammatory disease affecting 0.5%−1% of the world population. [72][73][74] If left untreated or ineffectively treated, rheumatoid arthritis typi-

| Tumour-associated bone destruction
Multiple myeloma is a destructive cancer that mainly occurs in bone marrow. 80 Studies have shown that fatty acids of different types play either pro-death or pro-survival roles in multiple myeloma. For example, PA could activate apoptosis in multiple myeloma cells and thereby serves as a potentially direct anti-myeloma strategy. 81 EPA and DHA could also initiate apoptosis and promote drug sensitivity in multiple myeloma cells, with a mechanism involving NF-κB inhibition concomitant with activation of mitochondrial defects leading to caspase-3 activation and apoptosis. 82 In addition, EPA and DHA modulate p53/miR-34a/Bcl-2 axis to enhance dexamethasone (Dex)-sensitivity in multiple myeloma cells where they trigger p53 expression and subsequent increase of miR-34a levels in U266 cells, and finally activate Bcl-2 to induce apoptosis of multiple myeloma cells. [83][84][85] By contrast, SFAs and ω-6 LCPUFAs represent energy sources for multiple myeloma cells, and ratio of ω-3/ω-6 fatty acid intake is critical for the maintenance of multiple myeloma cell survival. 86,87 Bone metastasis is a pernicious complication 88 occurring in virtually 60% of patients with osteolytic breast or osteogenic prostate cancers and at a smaller rate in patients with other cancer types. 89,90 Patients with bone metastasis suffer from severe pain, bone fracture and osteolytic lesions, which symptoms are primarily attributed to aberrant bone resorption mediated by osteoclasts. 91,92 In osteolytic metastasis mice model originating from MDA-MB-231 human breast cancer, researchers found that supplementation with DHA and EPAenriched fish oil prevented breast cancer metastasis-induced bone osteolysis, 93 suggesting potential therapeutic effects of fatty acids for osteolytic bone metastasis. In mechanism, both DHA and EPA reduce the mRNA and protein levels of CD44 in breast cancer cells to inhibit cancer invasion; moreover, compared to EPA, DHA has profound anti-inflammatory effects via inhibiting TNF-α secretion and NF-κB activation in macrophages and thus exhibits stronger suppression of osteoclast activity to attenuate the related osteolysis. 94 However, in osteogenic metastasis derived from prostate cancer, fatty acids such as AA could facilitate metastatic cancer cell implantation and propagation via preparation of bone microenvironment "soil" for cancer cells by activating bone marrow adipocyte formation, 95

| Other bone disorders
Fatty acids are also involved in non-typical skeletal diseases such as osteomyelitis, a bone inflammatory process initiated by infection of pyogenic organisms 97 that predominantly occurs in long bones of children, and in hips, feet, jaws and spine of adults. [98][99][100] This disease is characterized by severe damage to bone tissue and bone marrow, and probably accompanied by high morbidity and mortality. 100 Accumulating evidence has shown that ω-3 LCPUFAs could effectively combat microbial pathogenesis in osteomyelitis. [101][102][103] Furthermore, combination of vancomycin and ω-3 LCPUFAs has been suggested to be a reliable therapeutic strategy against S aureus-induced osteomyelitis, with a mechanism involving inflammation alleviation by reducing TNF-α and interleukin 6 (IL-6) levels as well as antioxidant activity by decreasing SOD activity. 97 Taken together, according to currently available pre-clinical experiments ( Table 2) and clinical studies (Table 3)

| Receptors involved in fatty acids-modulated bone metabolism
Cellular membrane-bound and nuclear receptors, such as G pro- MCFAs (C9-C14). 115-117 GPR120, which is expressed on osteoblasts and osteoclasts and could be stimulated by long-chain saturated (C14-C18) and long-chain unsaturated fatty acids (C16-C22), [109][110][111] has been shown to mediate the anti-inflammatory effects of DHA in macrophages. 118 And GRP120 could enhance ω-3 LCPUFAs-induced osteoblastic bone formation by inducing β-catenin activation and reduce osteoclastic bone resorption by suppressing NF-κB signalling, 14 and GPR120 could also modulate the bi-potential differentiation of BMMSC in a dose-dependent manner. 119 In addition to the

| Fatty acids and osteoblasts
Osteoblasts are mononuclear cells predominantly involved in bone

| Fatty acids as negative regulators of osteoblasts
Palmitate, a kind of LCSFAs, impedes osteoblast differentiation and induces cell death via lipotoxicity. 105 Palmitate could induce autophagy in osteoblasts dependent on Beclin and PI3K, 178 and autophagy serves as a protection mechanism in preserving osteoblasts from lipotoxicity. 179 Palmitate also promotes apoptosis of osteoblasts through both extrinsic and intrinsic pathways, and PA-  of ω-6/ω-3 LCPUFAs blocks PPAR-γ activation and thus enhancing F I G U R E 3 RANK/RANKL/OPG pathway in fatty acids-modulated bone metabolism. The well-documented RANKL signalling pathway exerts essential role in osteoclastogenesis. RANKL binds to RANK on the surface of osteoclast precursor cells and activates three distinct downstream signalling pathways. The MAPK pathways characterized by downstream factors ERK, p38 and JNK play pivotal role in cell death and survival. The NF-κB signalling pathway is activated following IκBα phosphorylation and degradation. The p50 and p65 subunits of NF-κB are released and translocated into the nucleus to activate the transcription of target genes. The PI3K/mTOR pathway is also activated upon binding of RANKL to RANK, which triggers the activation of PDK1s and Akt leading to the inhibition of the TSC complex and subsequent mTORC1 formation. The mTORC1 phosphorylates S6K1 as well as 4E-BP1, which further regulate protein synthesis, cell proliferation, angiogenesis and autophagy. However, mTORC2 acts as an essential modulator of actin cytoskeleton, cell survival and lipid metabolism. RANKL, receptor activator of nuclear kappa-β ligand; TGF-β, transforming growth factor β; JNK, c-jun NH2-terminal kinase; Akt, protein kinase B; S6Ks, S6 kinases; 4E-BP1, 4E-binding protein 1 osteoblastogenesis. 56 Besides, SCFAs such as butyrate promote osteoblast formation and differentiation by enhancing production of bone sialoprotein and osteopontin; moreover, it stimulates osteoblasts to secret OPG and thus facilitating the blocking of osteoclast differentiation. 185

Fatty acids derivatives
RvE1 is an EPA metabolite that is closely associated with inflammation-induced bone disorders. In IL-6-stimulated osteoblasts, supplement of RvE1 leads to significant disruption of PI3K-Akt pathway, which interacts with NF-κB, MAPK and p53 signalling to modulate protein synthesis, cell differentiation and apoptosis.
In inflammatory bone disorders, changes in production of pro-in-

| Fatty acids and osteoclasts
Osteoclasts are multinucleated giant cells with bone resorptive activity. Two essential factors secreted by osteoblasts, macrophage colony-stimulating factor (M-CSF) and RANKL, are responsible for osteoclast precursors proliferation and osteoclastogenesis.
Importantly, RANKL could prevent apoptosis of osteoclasts 3,4 and induce expression of osteoclast-specific markers and transcription factors such as nuclear factor of activated T cells c1(NFATc1). 4,6 As bone-resorbing cells, [192][193][194][195] osteoclasts highly express bone resorption-associated proteins including osteoclast-specific markers cathepsin K (CTSK), tartrate resistant acid phosphatase (TRAP) and matrix metalloproteinase 9 (MMP-9). 4,196 Specifically, CTSK breaks down organic components in bone, 197,198 TRAP is implicated in cell adhesion upon activation by CTSK, 199,200 and high levels of MMP-9 commonly occur in resorption lacunae. 201 Multiple fatty acids have been found to promote or suppress osteoclast activity, in most cases via regulation of RANKL signalling. Effects of fatty acids on osteoclast functions demonstrate their potential applications as therapeutic reagents against resorption-associated bone disorders such as osteoporosis and rheumatoid arthritis.

| Fatty acids as positive regulators of osteoclasts
Accumulating evidence has shown that PA enhances RANKLmediated differentiation of osteoclasts by upregulating expression levels of RANK; importantly, PA has been reported to be sufficient for osteoclast differentiation in conditions even without RANKL. 202

| Fatty acids as negative regulators of osteoclasts
LCPUFAs LCPUFAs such as DHA and AA could exert inhibitory effects on os-

| Fatty acids and BMMSCs
BMMSCs are multipotent cells characterized by surface markers of CD105, CD73, CD90, CD44, CD29 and CD146 9 with differential potentials into osteoblasts, chondroblasts and bone marrow adipocytes. 7  Oleate inhibits palmitate (palm)-induced apoptosis and increases BMMSCs proliferation. 27 Palm has been shown to induce lipotoxicity, whereas oleate fully neutralizes palm-induced lipotoxicity and pro-inflammatory response. Oleate exhibits cytoprotective effects by deactivating palm-induced pathways and fostering esterification of Palm into triglycerides. 226 More specifically, Ole inhibits palm-induced activation of ERK and NF-κB signalling, which results in pro-apoptotic effects in BMMSCs. 226,227 Also, decline in IL-6 and IL-8 expression and secretion levels by Ole treatment was also observed. 228 Furthermore, Ole maintains the oxidative levels of palmitate. 27 Hence, OA represents a potential therapeutic agent in combating PA-induced lipotoxicity in the bone.

| Fatty acids as negative regulators of BMMSCs
As mentioned above, palmitate triggers BMMSCs apoptosis and reduces their proliferation. 27

| Fatty acids and osteocytes
Osteocytes are osteoblast-derived cells located in lacunae surrounded by mineralized bone matrix, with the ability to support bone structure and receive machine sensation. Importantly, osteocytes can serve as endocrine cells to synthesize and express important regulatory molecules including RANKL, Dickkopf-1 (DKK1) and sclerostin (SOST) [233][234][235] and thus participating in bone resorption and formation regulation by coupling osteoclast and osteoblast activities. 6 Studies have shown that fatty acids such as PA and PGE2 have noteworthy influences on osteocyte metabolism, which might provide novel therapeutic strategies for bone diseases like osteoporosis.

| Fatty acids in osteocytes-mediated bone metabolism
PGE2 released by osteocytes are important regulators of bone formation. For example, PGE2 produced by low-intensity pulsed ultrasoundstimulated osteocytes could enhance osteoblasts differentiation but inhibit their proliferation in vitro. 236 In addition, mechanical loading or fluid flow shear stress on osteocytes can release PGE2 to regulate osteoblast proliferation and differentiation. 237 In mechanism, loading-in- Moreover, PGE2 could promote production of 8-nitro-cGMP in osteocytes to enhance osteoclasts differentiation. 240

| Fatty acids in osteocytes-associated bone disorders
Investigations have suggested that PA can cause lipotoxicity in osteocytes. PA results in apoptosis and inhibits survival in osteocytes by induction of autophagy failure, which is indicated by conspicuous increase in LC3-II and reduction of autophagosomes/lysosomes in cytoplasm. 234 In addition, PA exerts effects on bone turnover by decreasing expression of DKK1, RANKL and sclerostin in osteocytes. 234 Given osteocytes apoptosis and dysfunction are two common changes in osteoporotic bone, PA might play a part in the pathogenesis as well as potential therapeutic applications in osteoporosis. In addition, fatty acids oxidation can serve as energy source for osteocytes. 241 In vivo evidence has shown that fatty acid oxidation could compensate dysfunction of energy metabolism and osteocytes formation caused by glucose transporter-4 deficiency in osteoblasts and osteocytes of mice. 242 Importantly, activation of β-catenin regulated by Wnt-Lrp5 signalling affects oxidative potential and fatty acids utilization in osteocytes and thus is responsible for expression of key enzymes during fatty acid oxidation. 241 Therefore, fatty acid oxidation in osteocytes exerts regulatory effects on bone fat and body mass, which might have regulatory roles and therapeutic applications in metabolic disease-associated bone disorders.

SFA and its metabolites
Several studies have shown that animals fed with high-SFAs diet exhibit accelerated cartilage degeneration, 260  and thus induces apoptosis of chondrocytes. 272 Taken together, palmitate has potent therapeutic implications for inflammatory bone diseases such as osteoarthritis.

F I G U R E 4
Modulation of fatty acids on specific bone cell types. Multiple receptors for fatty acids including GPRs, ChemR23, TLRs and PPARs are found in pre-osteoclasts, mature osteoclasts, osteoblasts and chondrocytes. Several GPRs including GPR18, GPR41, GPR43 and GPR109A are receptors for SCFAs (C2-C5) expressed in both osteoclasts and osteoblasts. GPR40, found on osteoclasts, could be activated by medium/long-chain fatty acids with a chain length of C8-C22. GPR84, whose expression in macrophages and adipocytes could be enhanced under inflammatory conditions, is a receptor for MCFAs (C9-C14). GPR120 is expressed on osteoblasts and osteoclasts and could be stimulated by LCSFAs (C14-C18) and LCUFAs (C16-C22). PTH1R, belonging to GPR2 family, could be antagonized by ω-3 LCPUFAs to promote osteoblast activity. PPARs, with known ligands including LCPUFAs and metabolites such as PGE2, are nuclear receptors that regulate lipid metabolism by acting as transcription factors in BMMSCs, osteoblasts, osteoclasts and chondrocytes. TLRs, including TLR2 and TLR4, are found in osteoblasts, pre-osteoclasts, osteoclasts and chondrocytes. Their ligands are mainly SFAs and LCPUFAs and are involved in inflammatory action. ChemR23 can act as chemerin receptor as well as RvE1 receptor in bone tissue cells such as osteoclasts and osteoblasts. Interactions of fatty acids with specific receptors induce transduction of transmembrane specific signals and activation of various downstream signalling pathways including NF-κB, NFATc1 or Runx2-mediated transcriptional regulation, and further modulating bone microenvironment homeostasis and pathological bone remodelling. GPRs, G protein-coupled receptors; chemR, chemokine-like receptor; TLR, toll-like receptor; SCFAs, short-chain fatty acids; MCFAs, medium-chain fatty acids; LCSFAs, long-chain saturated fatty acids; LCUFAs, long-chain unsaturated fatty acids; PTH1R, parathyroid hormone type 1 receptor; LCPUFAs, long-chain polyunsaturated fatty acids; PPARs, peroxisome proliferator-activated receptors; PGE2, prostaglandin E2; SFAs, saturated fatty acids; RvE1, resolvin E1; NF-κB, nuclear factor-kappa B; NFATc1, nuclear factor of activated T-cell cytoplasmic 1; Runx2, runt-related transcription factor 2 ω-6 PUFAs and their metabolites A growing body of evidence has shown that a higher ratio of ω-6to-ω-3 PUFAs might exert negative influences on cartilage. 273 As for specific mechanisms, ω-6 PUFAs such as ALA and AA aggravate cartilage damage by serving as precursors for pro-inflammatory prostanoids, while ω-3 PUFAs such as EPA and DHA protect cartilage by being metabolized to anti-inflammatory mediators such as protectins and resolvins. 273,274 Moreover, AA-derived PGE2 could serve as important inflammatory mediator to regulate inflammatory reactions of chondrocytes. Studies have shown that PGE2 could suppress differentiation of chondrocytes by activating downstream receptors protein kinase A (PKA) and protein kinase C (PKC), which might be responsible for activation of transcription factors associated with collagen X production. 275 Taken together, fatty acids exert multiple effects on specific bone cell types and thereby associated bone diseases (Table 4), which might be mediated via distinct mechanisms at cellular and molecular levels ( Figure 4). Understanding the mechanistic implications of fatty acids in bone cells will greatly benefit their further utilization in related bone disorders.

| CON CLUDING REMARK S
In this review, we reviewed impacts of fatty acids on bone me- Taken together, we conclude that involvement of fatty acids in bone diseases pathogenesis might provide potential therapeutic targets for interventions of bone disorders, and promising fatty acids with therapeutic effects might be used directly or indirectly in nutritional or drug formulations for prevention and treatment of specific types of bone disorders.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Bao M and Zhang K gathered relevant literature and wrote the manuscript; Wei Y, Hua W and Gao Y interpreted data from pathological and experimental studies; and Li X and Ye L provided financial support, revised and reviewed the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.