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Overview of RAW264.7 for osteoclastogensis study: Phenotype and stimuli

Lingbo Kong, Wanli Smith, Dingjun Hao

Journal of Cellular and Molecular Medicine · 2019 · Review · CC BY

doi:10.1111/jcmm.14277

Abstract

Bone homeostasis is preserved by the balance of maintaining between the activity of osteogenesis and osteoclastogenesis. However, investigations for the osteoclastogenesis were hampered by considerable difficulties associated with isolating and culturing osteoclast in vivo. As the alternative, stimuli‐induced osteoclasts formation from RAW264.7 cells (RAW‐OCs) have gain its importance for extensively osteoclastogenic study of bone diseases, such as rheumatoid arthritis, osteoporosis, osteolysis and periodontitis. However, considering the RAW‐OCs have not yet been well‐characterized and RAW264.7 cells are polymorphic because of a diverse phenotype of the individual cells comprising this cell linage, and different fate associated with various stimuli contributions. Thus, in present study, we provide an overview for current knowledge of the phenotype of RAW264.7 cells, as well as the current understanding of the complicated interactions between various stimuli and RAW‐OCs in the light of the recent progress.

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From PubMed Central, U.S. National Library of Medicine, as current on Oct 22, 2026. Paragraph numbers are ours, so an answer can point at one.

INTRODUCTION

The integrity of bone and skeleton tissue is preserved by the balance maintaining between the activity of the osteoblasts conducted bone forming and osteoclasts conducted bone resorbing, which ensures no net change in bone mass.1, 2 However, the balance was constantly cycled for remodelling bone tissue with the starting from the osteoclasts formation (osteoclastogenesis),3 and investigations for the osteoclastogenesis were hampered by considerable difficulties associated with isolating and culturing osteoclast in vitro.4 To solve the issues of osteoclast differentiation in vitro, initial studies try to differentiate osteoclasts through co‐culturing various cell types, such as splenocyte precursor cells, primary monocytes/macrophages with primary osteoblasts or stromal cell linages,5, 6 which subsequently followed the collagenase treatment, to allow releasing of the osteoblast/stromal cell component, finally leading to the formation of osteoclastic cells.7 However, except few cell linages could be used for decipher the mechanisms involved in osteogenesis and bone homeostasis, the most cell linages finally be proved as an invaluable research cellular tool for study osteoclastogenesis.4

Osteoclasts arise in the bone marrow from the fusion of haematopoietic cells of a monocyte/macrophage lineage after stimulation by macrophage colony‐stimulating factor (M‐CSF)8 and receptor activator of nuclear factor‐κB ligand (RANKL).9 RANKL acts directly on osteoclast precursors, via the receptor RANK, to induce differentiation of precursors to multinuclear bone resorbing cells.10 As the consequence of the discoveries and extensively exploring for M‐CSF and RANKL, study for osteoclasts in the fields of cellular developing, functional activity and biological molecular mechanism stepped into new era.11, 12, 13 Primary osteoclast precursor cells, which including bone marrow macrophage (BMMs),14 splenocytes5 and peripheral blood monocytes,15, 16 can now be induced to osteoclastic differentiation in vitro by culturing in the presence of recombinant M‐CSF and RANKL.17, 18 However, the use of these primary cells raised difficulties, which associated with these cell linage characters, such limits including the availability and variation in response patterns among different cellular study preparations.19, 20 Moreover, as a genetic virtually untransfectable cell linage, primary cell‐derived osteoclasts and their precursors are poorly suited for genetic manipulation and promoter studies.20

In addition to primary cells, the RAW264.7 cells are monocyte/macrophage like cell linage, originating from Abelson leukemia virus transformed cell linage derived from BALB/c mice.21 Initially, RAW264.7 cells have been described as an appropriate model of macrophages, as the cell are capable for performing pinocytosis and phagocytosis.21, 22 Later, further studies proved RAW264.7 cells could respond to stimuli in vitro and subsequently generate multinucleated cells with the hallmark characteristics expected for fully differentiated osteoclasts (RAW‐OCs).23 RAW‐OCs have been extensively employed in studies of osteoclastogenesis for more than 20 years. In fact, as the pioneers of this field Collin‐Osdoby and Osdoby pointed out, in parallel or as a prelude to primary cells induced osteoclasts, such as bone marrow macrophages derived osteoclast (BMM‐OCs), RAW‐OCs presenting many advantages of osteoclastogenic cellular model system over the BMM‐OC populations, mainly including (a) easy culture and passage; (b) widespread availability of this cell line to most researchers; (c) homogeneous nature of the osteoclast precursor populations (devoid of osteoblast, stromal, lymphocytes, or other cell types; (d) close correlation in characteristics, gene expression, signalling and developmental or functional processes among the RAW‐OCs, primary precursor cell linage derived osteoclasts and isolated in vivo formed osteoclasts.4 Therefore, RAW‐OCs can be used for studying osteoclastogenesis through different methods for various study purposes, including: biochemical, immunological, physiological, molecular and functional assays according to various study procedures.

RAW264.7 cell linage is well‐characterized with regard to macrophage‐mediated immune, metabolic and phagocytic functions21 and is increasingly used and accepted as a cellular model of osteoclastogenic study;24, 25, 26 however, with the vastly usage of RAW‐OCs for understanding the osteocalstogenesis in the past two decades, there raised considerable requirements for the extensively understanding the RAW‐OCs and associated cellular biological mechanisms.27, 28 On the other hand, document studies reported that during the osteoclastic induction, various stimuli might lead different cellular fates during the RAW‐OC induction.29, 30, 31 Considering RAW264.7 cell‐derived osteoclasts have not yet been well‐characterized, and RAW264.7 cells are polymorphic with respect to the phenotype of the individual cells comprising this cell line and different results associated with various stimuli contributions.32, 33, 34, 35 Therefore, in our present review, we will summarize the current knowledge of the phenotype of RAW264.7 cells and the current understanding of complicated interactions between various stimuli and RAW‐OCs in the light of the recent progress in this field.

PHENOTYPE STUDY OF RAW264.7 AND ITS ROLE ON THE REGULATION OF RAW‐OCS

Macrophages are very sensitive to environmental conditions, specifically, in response to different stimuli signals, macrophages (M0 or Mφ)36 can display different functional phenotypes including classically activated (M1 or pro‐inflammatory) and alternatively activated (M2 or anti‐inflammatory) phenotypes.37, 38, 39 Established RAW264.7 cells as an immortalized monocyte/macrophage cell lineage,40, 41 its phenotype might change with the passages and micro‐environments of the cell;32, 42 therefore, there raise a cautious for interpretation of data obtained from experiments conducted only on the established cell lines, which also including RAW‐OCs. On the other hand, their stability between various laboratories and passages is questionable.43 American Type Culture Collection, the main supplier of the cell lines, recommends the passage using are controlled above for passage No. 18, since study showing the induction efficacy might decrease with passage and consequently phenotype changing.4, 19, 32 Otherwise, previous study showed the elder RAW264.7 cells could change its morphology and decrease the production of proteins, which lead RAW264.7 cells resistant for differentiation and transduction.44 However, there still lack evidence for rigid classification criteria of cell lines between various passages and it seems to be cell‐linage dependent. Therefore, combined with previous excellent and novel studies in these fields,4, 32 in our experiences, it might be a reason for different RAW‐OC induction efficacies among various study groups. Therefore, confirmation of RAW264.7 stability through phenotype study among is important for the proper further data interpretation.32

Collin‐Osdoby et al4 have firstly reported that the recommendation of RAW264.7 cell line up to passage No. 18, due to decreasing efficacy for RAW‐OCs production. To clarify the phenotype stability of RAW264.7 macrophages, Taciak et al32 performed systematic analyses of RAW264.7 gene expression with a panel of 28 gene, which contained with macrophage metabolisms. Their study results manifested that through the passages the expression of genes in RAW264.7 from passage No. 5 to No. 50 should be classified into three expression sub‐panels, which include (a) increasing expression; (b) stable expression; and (c) fluctuating expression.32 Specifically, the genes from subpanel‐1 are involved in macrophage functions (including: CD11b, CD14, Ireb‐2, TfR (transferrin receptor), CD36, iNOS, CD11c, VEGFR2 (vascular endothelial growth factor receptor 2), TRAP (tartrate‐resistant acidic phosphatase), TIM‐2 (T‐cell immunoglobulin and mucin domain 2) and HIF‐2α (hypoxia inducible factor‐2α)), which demonstrated a significantly stable from passage No. 5 to No. 50.32 However, comparing to RAW264.7, the bone marrow macrophage‐M0 (BMM‐Mφ) has been reported markedly express CD169.45, 46, 47 Otherwise, the CD11c and iNOS in subpanel‐1 are demonstrated as highly expressed in pro‐inflammatory macrophage stage (M1).48

Similarly, genes in subpanel‐2 and subpanel‐3 also associated with macrophage activation including: CD86, HIF‐1α, CD11a, CD18, CD206, CD200R, Glut1 (Glucose transporter 1) and Ly6c and TfR2, Arg1 and SCARA‐5b (scavenger receptor class A member 5) respectively.32 Interestingly, however, as an iNOS inhibitory genes, the Arg1 also highly expressed in another polarized macrophage stage: anti‐inflammatory (M2) phenotype,49, 50 and its expression in RAW264.7 has be showed significantly higher in the passage of No. 15.32 Notably, the expression of TRAP, which involved in osteoclastogenesis and as one of osteoclastic specific genes remains stable in RAW264.7 cell linage from the passage No. 5.32 Besides that, Wang et al51 have reported that CD109, as a glycosyl‐phosphatidylinositol (GPI)‐anchored protein play a crucial role in RANKL‐induced RAW‐OCs formation and bone resorptive function. The study suggested that CD109 might be a co‐receptor or decoy receptor for TGF‐β that would otherwise bind to cell‐associated TGF‐β receptors, when TGF‐β signalling is down‐regulated by CD109, subsequent inhibition SMAD family member 3 (SMAD3) signalling occurs which might lead to a decrease in the formation of TRAF6 promoted of formation of TAK1 complexes, which comprise the TAK1 catalytic subunit (TAK1), TAK1‐binding protein 1 (TAB1), finally could inhibit RAW‐OCs formation51 (Figure 1).

RANKL AND ITS ROLE IN RAW‐OCS INDUCTION

Receptor activator of nuclear factor kappa B (RANK also known as ODF, OPGL and TRANCE), which is one of TNF receptor family members, is expressed in osteoclasts and their precursor cells as the receptor of RANKL.3, 9, 10, 52 Downstream signalling through RANK is essential for osteoclastogenesis.18, 19 RAW264.7 cell linage has been identified as a transfectable RANK expressing cell linage.53, 54, 55 Hsu et al56 firstly reported RANKL induced osteoclastogenic RAW‐OCs. Partly, similar to its counterpart BMM‐OCs, RAW‐OCs derived from the RANKL/RANK binding and NF‐κB activiation, consequently further presented a significant bone resorptive function in vitro.57, 58 Since then, RAW‐OCs have been extensively employed in osteoclastogenesis studies for more than 20 years.

RANKL‐induced RAW‐OCs have been proved as an important cellular model for bone homeostasis studies because the merits of RAW264.7 cell linage, which lies in its widespread availability, homogeneous nature of primary osteoclasts population (devoid of osteoblasts, lymphocytes and stroma, etc), and ease of culture and transfection for genetic manipulation.20, 27, 28, 59, 60, 61 Otherwise, osteoclast differentiation is dependent upon the intimate cellular interaction of myeloid preosteoclast precursors with either osteoblasts or stromal cells and is influenced by a wide range of local factors.12, 14, 62, 63, 64 Whereas, RANKL, expressed on both stromal cells and osteoblasts, plays an essential role in the regulation of osteoclast differentiation.10, 65 Thus, a soluble recombinant form of RANKL (sRANKL) is sufficient to replace fully the requirement for osteoblast and stromal cell interactions in the induction of osteoclast differentiation in in vitro culture system.66, 67 However, despite the advantages of RANKL for its role in the RAW‐OCs stimulation, there still exist conspiracies during the induction of RANKL‐induced osteoclast from RAW264.7. For instance, l‐glutamine for decades has been recognized as an important factor for cell line growth and differentiation in in vitro culture systems,70, 71 otherwise the importance of l‐glutamine in osteoclastic cellular culture medium was demonstrated by Indo et al73 However, recently Nguyen et al74 have reported that during the RANKL stimulated RAW‐OCs induction media, the plus with l‐glutamine at the concentration 4‐6 mM could decrease the total number of multinucleated RAW‐OCs, which could reverse by reducing the concentration of l‐glutamine to 1‐2 mM. Although these results might be because of the accumulation of ammonia/ammonium ion from the breaking down of l‐glutamine in the culture system,75 it definitely raised requirements for comprehensive understanding the RANKL for its role in RAW‐OCs induction, which include the efficacy of induction, relevant cellular mechanisms and association of metabolic products.

Besides that, Cuetara et al20 have reported the osteoclastogenic activity is various among each colony of RAW‐OCs, even in the same passage and same culture condition. Interestingly, however, their study found that all RAW264.7 clones tested expressed the RANKL receptor RANK and expressed the osteoclast marker genes TRAP and cathepsin‐K mRNA with RANKL treatment.20 Comparing BMMs, RAW264.7 cell linage are a transformed macrophage like cell line derived from the lymphoma of a male BALB/c mouse infected by the A‐muLV.76 The retrovirus encodes an oncogenic form of the Abelson kinase, v‐Abl, which is a fusion protein where portions of the retroviral Gag protein substitute regions of the SH3 domain of c‐Abl, rendering the tyrosine kinase constitutively active.77, 78 Moreover, previous study reported RANKL induced RAW‐OC expressed β3 integrin mRNA,20, 81 which substitute the regions of SH2 domain.82, 83 Therefore, the cellular mechanisms of RANKL‐induced RAW‐OCs formation and the bone resorptive activity of RAW‐OC may occur through various signalling cascades cross‐talking, which is the discrepancy in RANKL‐induced BMM‐OCs formation. Specifically, different to BMM‐OCs, document reports have shown that RAW 264.7 cells can proliferate and undergo osteoclastogenic differentiation independently from M‐CSF.28, 85 The underlying cellular mechanisms, might lie in v‐Abl in RAW 264.7 cells, were limited to M‐CSF‐related signalling pathways. Although these results raise a cautious consideration for using RAW‐OCs in the studying signalling downstream of M‐CSF cascades, it is still acceptable for studying the downstream of RANKL (Figure 2).

RANKL binding with RANK induces the recruitment of tumour necrosis factor receptor‐associated factors (TRAFs) to the cytoplasmic domain of RANK, including TRAF‐2, TRAF‐5 and TRAF‐6.86, 87, 88 This engagement leads to the activation of a signalling cascade with downstream targets, which including extracellular regulated kinase (ERK),81 p38 mitogen‐activated protein kinase (p38),59 c‐Jun N‐terminal kinase (JNK),56, 89 phosphatidylinositol‐3 kinase (PI3K)90 and IκB kinase.91, 92 Consequently, crucial osteoclastogenic transcription factors, such as activator protein‐1 (AP‐1:c‐Fos and c‐Jun),93 nuclear factor‐κB (NF‐κB),94 and nuclear factor of activated T cells c1 (NFATc1) are activated.27 In that, NFAT family members are previously been reported expressed in RAW264.7 cells and that their expression is up‐regulated in response to RANKL stimulation.95, 96 In fact, calcineurin inhibitory peptide could inhibit the RANKL‐induced RAW264.7 monocyte/macrophage cell line into mature RAW‐OCs. Otherwise, ectopic expression of a constitutively active calcineurin‐independent NFATc1 mutant in RAW264.7 cells fused into morphologically giant multinucleated RAW‐OCs, which possess the capabilities for the resorption of a physiological mineralized matrix substrate.97, 98, 99 Meanwhile, as critical transcriptional factors, c‐Fos and NFATc1 are sufficient to induce the expression of osteoclast‐specific genes expression, including TRAP and cathepsin K,100 and fusion‐specific genes expression, which including dendritic cell‐specific transmembrane protein (DC‐STAMP).101 However, although initial studies speculated that RANKL/RANK signalling is indispensable for RAW‐OCs formation, extensive study explored the understanding of the contingent series of signalling events involved in RAW‐OCs is far from completely understood.

LIPOPOLYSACCHARIDE AND ITS ROLE IN RAW‐OCS INDUCTION

Physiological defense responding is important for the host to survive infection.76, 102 Lipopolysaccharide (LPS) as a major component of the outer membrane of Gram‐negative bacterial factor for inflammation has ability to induce the expression of a variety of pro‐inflammatory cytokines and nitric oxide producing enzyme iNOS in macrophage.30, 103 LPS or the consequent various cytokines elicited in infectious lesions may modulate physiological and pathological osteoclastogenesis respectively. However, further lead to a pathological bone resorptive condition.

RAW264.7 is one of the three cloned cell lines (RAW309 and WR19M), which have been established from murine tumours induced with Abelson leukemia virus and express properties of macrophages.76 Macrophages (Mφ or M0) are inflammatory cells with high capacity for engulfing and digesting pathogens and cell debris.104 Otherwise, based on the micro‐environment, macrophages play increasingly defined roles in orchestrating the healing of various damaged tissues and show high heterogeneity, plasticity and adaptation abilities.105, 106 Specifically, during the inflammatory osteoclastogenesis, in response to multiple signals or cytokines, macrophages might differentiate into different types of multinucleated cells to internalize the large amounts of un‐necessaries such as strong inflammatory induced host cells, and wear debris, which originally from orthopedic instruments.104 During this process, macrophage changing their morphologies by cellular fusion, further formed into multinucleated giant cells (MGCs), which bear the function for engulf and digestion.104 In fact, osteoclasts are one of MGC cells, which are attached tightly to the bone surface, and secrete protons and lysosomal enzymes for bone resorption.

Initially, LPS has only been suggested to promote the differentiation and survival of BMM‐OCs through generating kinds cytokines, such as PGE2, IL‐1, RANKL and TNF‐α.107, 108 Interestingly, although previous studies have demonstrated RANKL and M‐CSF are essential and sufficient for BMM‐OCs induction,85, 109 recent studies have suggested that M‐CSF might not participate in the LPS‐induced RAW‐OCs in vitro culturing system.28, 30 This speculation proved by the anti‐M‐CSF antibody could not inhibit LPS‐induced RAW‐OCs formation and M‐CSF could not be detected in the supernatant from cultures of LPS treated RAW264.7 cells, besides pretreatment with M‐CSF has not shown evidence for enhancing the RAW‐OCs formation. Otherwise, RANKL do not seem to be involved in LPS‐induced osteoclast formation from RAW264.7 either. Islam et al110 firstly demonstrated that LPS causes the formation of TRAP‐positive MGC in RAW264.7 cells and that they exhibit a pit‐forming activity on calcium carbonate‐coated plates. These results suggested that RAW264.7 cells might act as osteoclast progenitors and could differentiate to osteoclasts in response to LPS. Meanwhile, Hotokezaka et al111 have reported LPS‐induced RAW264.7 cells fusion and osteoclastic RAW‐OCs formation by RANKL‐independent manner. However, the molecular mechanisms underlying the LPS‐induced RAW‐OC formation still remain unclear. One speculation is that LPS might mimic RANKL‐induced osteoclast formation via activation of NF‐κB and SAPK/JNK, because the inhibition of SAPK/JNK by the selective inhibitor (SP600125) could inhibit LPS‐induced osteoclastogenesis112 (Figure 2).

Interestingly, on the contrary, Nakanishi‐Matsui et al113 reported that LPS‐induced multinuclear cells did not express osteoclast‐specific enzymes including TRAP and cathepsin K. The reason for their speculation lying in the period of osteoclast formation is observed at third to seventh day after RANKL induction in RAW264.7. Whereas, the multinucleated cells derived from RAW264.7 cells are formed within 16 hr after LPS induction.113 However, this rapid formation should be reasonable because the response to LPS should correspond to that to bacterial infection.113 Moreover, LPS‐induced RAW‐OCs formation does not require the assistance of other cells, whereas primary osteoclast precursors require RANKL, which secreted from osteoblasts. Otherwise, although RAW264.7 cell linage has been identified as a transfectable RANK expressing, the LPS‐induced RAW‐OC was associated with RANKL‐independent signalling cascades.111 Besides that, even in Nakanishi‐Matsui study, they found these LPS‐induced giant multinucleated cells manifested as a promising internalization ability for polystyrene beads (diameter 6‐15 μm),113 this cellular activities similar to the osteoclasts were induced by periprosthetic wear debris. Further, they found the internalizing efficacy of LPS‐induced multinucleated cells is better for osteoclast.

Studies, including ourselves previous report, stimuli could induce RAW264.7 fuse into giant sized RAW‐OCs differentiate.28, 113 In fact, depending on the species, osteoclasts involved in normal bone remodelling contain an average of 3‐10 nuclei.114 Based on this, conventional osteoclast could be defined as TRAP‐positive stained nucleus number greater than 3. Interestingly, however, according to the number of nucleus, osteoclasts could be classified into ‘big osteoclast’ and ‘small osteoclast’, which containing the nuclei number greater than 10 or among 2‐5 in each osteoclast respectively115 (Figure 3). Pathological study demonstrated that the large osteoclast dominated in inflammatory induced bone loss, which leads to extensively bone resorption, such as Paget's disease and periodontal diseases. Piper et al114 have demonstrated a decrease in volume of resorption per nucleus as the number of nuclei per cell increased. Moreover, other study demonstrated that approximately 40% of the large osteoclastic multinucleated cells were actively resorbing in the population while only 6% of the small osteoclastic multinucleated cells were doing likewise. Lees et al116 have showed the resorptive activity associated with intracellular pH, which presented lower level in the large and functional osteoclasts comparing to small and non‐resorptive osteoclasts, respectively. Otherwise, Manolson et al117 showed that ‘α3’ V‐ATPase, as the critical enzyme of osteoclast PH regulator, expression was significantly increased in the larger RAW‐OCs compared to smaller RAW‐OCs.118

In fact, LPS presented a dual modulating effect on BMM‐OCs induction. Zou et al119 showed LPS has not exhibit osteoclastogenic activity on BMMs, on contrary, their study has shown LPS inhibited RANKL‐induced osteoclast differentiation in the absence of stromal cells or osteoblast. Otherwise, our previous study showed pretreatment with M‐CSF and LPS has not shown any up‐regulatory effects on lower concentration of RANKL‐induced RAW‐OCs.28 These results consistently with one previous study results that LPS inhibits the osteoclast formation in whole bone marrow cells with 1,25‐dihydroxyvitamin D3 via GM‐CSF production.120 However, previous study speculated that GM‐CSF might play a crucial factor for suppressing the LPS‐induced BMM‐OCs formation.121

However, as a critical potential inducer, LPS could enhance the osteoclastogenesis in RANKL‐pretreated BMMs,122 even if present in the absence of exogenous RANKL. Document interesting studies reported that LPS enhances osteoclast formation in cultures of whole bone marrow cells with dexamethasone and markedly enhances it in the presence of 1,25‐dihydroxyvitamin D3 and dexamethasone.123, 124 Otherwise, LPS stimulates osteoclastic bone resorption in vivo and in vitro in organ culture and increased osteoclast differentiation in whole bone marrow cell culture.30 Although, molecular study showed LPS does not exhibit osteoclastogenic property in the absence of osteoblasts or stromal cells, LPS could indirectly involved in promoting osteoclastogenesis. In that, LPS enhance RANKL expression in osteoblasts through Toll‐like receptors (TLRs),125 meanwhile LPS stimulates various cytokines secreting in micro‐environment, including IL‐1, PGE2 and TNF‐α, which involved in LPS‐mediated bone resorption.126 Interestingly, however, on the contrary to their counterpart, LPS‐induced RAW‐OCs formation in different fashion. Despite the aforementioned NF‐κB and SAPK/JNK signalling, LPS could up‐regulate MAPK and COX‐2 in RAW264.7 cells,30 which are the downstream signalling of RANKL/RANK. Therefore, LPS regulates the osteoclast formation in a complicated fashion, which presenting discrepancies among BMM‐OCs and RAW‐OCs induction, and associated molecular signalling. Therefore, depending on the experimental design and various cell lineage choice criteria, LPS could manifest an inhibitory effect or presenting induces on osteoclastogenesis.

TNF‐α AND ITS ROLE IN RAW‐OCS INDUCTION

TNF‐α, which is produced by many types of cells including monocytes and macrophages, has been proved to involve in bone resorption, particularly in inflammatory bone diseases such as rheumatoid arthritis127 and aseptic periprosthetic osteolysis.128 TNF‐α have been speculated to act directly by enhancing proliferation and activity of cells in the osteoclast lineage, or indirectly by affecting the production of osteoclast differentiation factors via osteoblast/stromal cells such as RANKL and its soluble decoy receptor, osteoprotegerin (OPG).111, 129

Despite the regulatory effects of TNF‐α on RANKL‐induced BMM‐OCs formation, recent studies focused on examining whether TNF‐α can promote osteoclastogenesis independently of RANKL signalling for both BMMs and RAW264.7 cell linages.111 However, different results among various research groups, for instance, has been reported that TNF‐α could promote osteoclast formation in vitro despite the RANKL signalling blockade,130, 131 while other reports have demonstrated that RANKL priming in osteoclast precursors is necessary for TNF‐α‐induced osteoclastogenesis.130 Further, TNF‐α is not necessarily required for osteoclastogenesis, erosive arthritis or osteolysis, as all these events could occur in the absence of TNF‐α. Therefore, these discrepancies reflected complicate molecular fashion during osteoclastogenesis, which involved multiple signalling cascades cross‐talking, cellular genetic backgrounds and various osteoclastogenic differentiate stages or culture conditions.132, 133 Thus, the relevance between RANKL and direct/indirect TNF‐α action in osteoclastogenesis remains unclear.

Nakao et al134 found that RANKL induces TNF‐α mRNA expression and secretion of TNF‐α protein in both spleen‐cell derived osteoclasts precursors (125 pg/mL) and RAW264.7 cells (600 pg/mL). However, this amount of TNF‐α (0.1‐1.0 ng/mL) by itself did not induce osteoclastogenesis in the absence of RANKL. They suggested that TNF‐α serves as an autocrine factor for osteoclastogenesis in cooperation with RANKL.135 Specifically, Zou et al136 explained this autocrine manner as RANKL induction of osteoclastogenesis is accompanied by a rapid and transient increase in TNF‐α mRNA abundance in the precursor cell; specific anti‐ TNF‐α antibodies or antibodies directed against TNF receptor type I inhibit the osteoclastogenic activity of RANKL in RAW264.7 cells. Therefore, it is reasonable for the speculation that TNF‐α induced RAW‐OC cross‐talking with RANKL signalling cascades. In fact, AU‐rich elements (AREs) of 3′ untranslated region (3′‐UTR), as cis‐element, involved in the regulation of TNF‐α mRNA transcription.137, 138 Otherwise, 3′‐UTRs associated with p38 MAPK/SAPK2 cascade induced the c‐Fos mRNA expression,139, 140 which is a crucial transcriptional factor of osteoclastogenesis. Moreover, p38 MAPK signalling pathways is also implicated in mediating RANKL‐induced osteoclastogenesis, suggested the increased TNF‐α mRNA abundance induced by RANKL is mediated by activating this signalling cascade leading to TNF‐α transcriptional stabilities. Besides that, most recently, Shinohara et al141 have reported that double‐stranded RNA‐dependent protein kinase (PKR) association is necessary for TNF‐a induced RAW‐OCs formation by p38MAPK, and ERK signalling (Figure 2).

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All animal care and experimental procedures were approved by Animal Care Committee of Hong‐Hui Hospital, Xi'an Jiaotong University College of Medicine (Animal Ethics Approval #1002017019) and conducted strictly followed by ‘the institutional guidelines for the care and use of laboratory animals at the Jiaotong University College of Medicine’.

CONSENT FOR PUBLICATION

The manuscript is approved by all authors for publication.

AVAILABILITY OF DATA AND MATERIALS

All data and materials were included in the manuscript.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS

WS, LK: conception and design, analysis and interpretation of data; draft the manuscript and revise it critically for important intellectual content; final approval of the version to be published. LK, DH: acquisition of data, analysis and interpretation of data; LK: conception and design, revise the manuscript critically for important intellectual content, final approval of the version to be published, account for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All the authors read and approved the final manuscript.

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    Not in our library10.1002/1521-4141(200204)32:4<1090::aid-immu1090>3.0.co;2-p

  19. 19.

    Collin‐Osdoby P, Yu X, Zheng H, Osdoby P. RANKL‐mediated osteoclast formation from murine RAW 264.7 cells. Methods Mol Med. 2003;80:153‐166.

    Not in our library10.1385/1-59259-366-6:153

  20. 20.

    Cuetara BL, Crotti TN, O'Donoghue AJ, McHugh KP. Cloning and characterization of osteoclast precursors from the RAW264.7 cell line. Vitro Cell Dev Biol Anim. 2006;42(7):182‐188.

    Not in our library10.1290/0510075.1

  21. 21.
    Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line · 2008
    No notice
  22. 22.

    Choi SY, Choi JY, Lee JM, Lee S, Cho, EJ. Tartary buckwheat on nitric oxide‐induced inflammation in RAW264.7 macrophage cells. Food Funct. 2015;6(8):2664‐2670.

    Not in our library10.1039/c5fo00639b

  23. 23.

    Xu XY, Guo C, Yan YX, et al. Differential effects of mechanical strain on osteoclastogenesis and osteoclast‐related gene expression in RAW264.7 cells. Mol Med Rep. 2012;6(2):409‐415.

    Not in our library10.3892/mmr.2012.908

  24. 24.

    Visagie A, Kasonga A, Deepak V, et al. Commercial honeybush (Cyclopia spp.) tea extract inhibits osteoclast formation and bone resorption in RAW264.7 murine macrophages‐an in vitro study. Int J Environ Res Public Health. 2015;12(11):13779‐13793.

    Not in our library10.3390/ijerph121113779

  25. 25.

    Zhao N, Tsuda H, Murofushi T, et al. Chaetocin inhibits RANKL‐induced osteoclast differentiation through reduction of Blimp1 in Raw264.7 cells. Life Sci. 2015;143:3077‐7.

    Not in our library10.1016/j.lfs.2015.10.027

  26. 26.

    Cao X, Kong X, Zhou Y, Lan L, Luo L, Yin Z. Glutathione S‐transferase P1 suppresses iNOS protein stability in RAW264.7 macrophage‐like cells after LPS stimulation. Free Radic Res. 2015;49(12):1438‐1448.

    Not in our library10.3109/10715762.2015.1085978

  27. 27.

    Crotti TN, Flannery M, Walsh NC, Fleming JD, Goldring SR, McHugh KP. NFATc1 regulation of the human beta3 integrin promoter in osteoclast differentiation. Gene. 2006;372:92‐102.

    Not in our library10.1016/j.gene.2005.12.012

  28. 28.

    Song C, Yang X, Lei Y, et al. Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells. J Cell Physiol. 2018.

    Not in our library10.1002/jcp.27852

  29. 29.

    Wu SH, Zhong ZM, Chen JT. Low‐magnitude high‐frequency vibration inhibits RANKL‐induced osteoclast differentiation of RAW264.7 cells. Int J Med Sci. 2012;9(9):801‐807.

    Not in our library10.7150/ijms.4838

  30. 30.

    Hou GQ, Guo C, Song GH, et al. Lipopolysaccharide (LPS) promotes osteoclast differentiation and activation by enhancing the MAPK pathway and COX‐2 expression in RAW264.7 cells. Int J Mol Med. 2013;32(2):503‐510.

    Not in our library10.3892/ijmm.2013.1406

  31. 31.

    Luo D, Kang L, Ma Y, et al. Effects and mechanisms of 8‐prenylnaringenin on osteoblast MC3T3‐E1 and osteoclast‐like cells RAW264.7. Food Sci Nutr. 2014;2(4):341‐350.

    Not in our library10.1002/fsn3.109

  32. 32.
    Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages · 2018
    No notice
  33. 33.

    Chen S, Lu Z, Wang F, Wang Y. Cathelicidin‐WA polarizes E. coli K88‐induced M1 macrophage to M2‐like macrophage in RAW264.7 cells. Int Immunopharmacol. 2018;54:52‐59.

    Not in our library10.1016/j.intimp.2017.10.013

  34. 34.

    Shi XD, Chen LW, Li SW, et al. The observed difference of RAW264.7 macrophage phenotype on mineralized collagen and hydroxyapatite. Biomed Mater. 2018;13(4):041001.

    Not in our library10.1088/1748-605x/aab523

  35. 35.

    Yamaguchi T, Movila A, Kataoka S, et al. Proinflammatory M1 macrophages inhibit RANKL‐induced osteoclastogenesis. Infect Immun. 2016;84(10):2802‐2812.

    Not in our library10.1128/iai.00461-16

  36. 36.

    Gabrusiewicz K, Rodriguez B, Wei J, et al. Glioblastoma‐infiltrated innate immune cells resemble M0 macrophage phenotype. JCI Insight. 2016;1(2).

    Not in our library10.1172/jci.insight.85841

  37. 37.
    Macrophage imbalance (M1 vs. M2) and upregulation of mast cells in wall of ruptured human cerebral aneurysms: preliminary results · 2012
    No notice
  38. 38.
    Macrophage Polarisation: an Immunohistochemical Approach for Identifying M1 and M2 Macrophages · 2013
    No notice
  39. 39.

    Garg K, Pullen NA, Oskeritzian CA, Ryan JJ, Bowlin GL. Macrophage functional polarization (M1/M2) in response to varying fiber and pore dimensions of electrospun scaffolds. Biomaterials. 2013;34(18):4439‐4451.

    Not in our library10.1016/j.biomaterials.2013.02.065

  40. 40.

    Lv R, Bao Q, Li Y. Regulation of M1type and M2type macrophage polarization in RAW264.7 cells by Galectin9. Mol Med Rep. 2017;16(6):9111‐9119.

    Not in our library10.3892/mmr.2017.7719

  41. 41.

    Pang X, Bian H, Su M, et al. Photoelectrochemical cytosensing of RAW264.7 macrophage cells based on a TiO2 Nanoneedls@MoO3 array. Anal Chem. 2017;89(15):7950‐7957.

    Not in our library10.1021/acs.analchem.7b01038

  42. 42.

    Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 2014;6:13.

    Not in our library10.12703/p6-13

  43. 43.

    Masters JR. Human cancer cell lines: fact and fantasy. Nat Rev Mol Cell Biol. 2000;1(3):233‐236.

    Not in our library10.1038/35043102

  44. 44.

    Wenger SL, Senft JR, Sargent LM, Bamezai R, Bairwa N, Grant SG. Comparison of established cell lines at different passages by karyotype and comparative genomic hybridization. Biosci Rep. 2004;24(6):631‐639.

    Not in our library10.1007/s10540-005-2797-5

  45. 45.

    Chow A, Lucas D, Hidalgo A, et al. Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J Exp Med. 2011;208(2):261‐271.

    Not in our library10.1084/jem.20101688

  46. 46.

    Wu AC, He Y, Broomfield A, et al. CD169(+) macrophages mediate pathological formation of woven bone in skeletal lesions of prostate cancer. J Pathol. 2016;239(2):218‐230.

    Not in our library10.1002/path.4718

  47. 47.

    Batoon L, Millard SM, Wullschleger ME, et al. CD169(+) macrophages are critical for osteoblast maintenance and promote intramembranous and endochondral ossification during bone repair. Biomaterials. 2017;196:51‐66.

    Not in our library10.1016/j.biomaterials.2017.10.033

  48. 48.

    Ono Y, Nagai M, Yoshino O, et al. CD11c+ M1‐like macrophages (MPhis) but not CD206+ M2‐like MPhi are involved in folliculogenesis in mice ovary. Sci Rep. 2018;8(1):8171.

    Not in our library10.1038/s41598-018-25837-3

  49. 49.

    Lewis KE, Rasmussen AL, Bennett W, et al. Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase. J Neuroinflammation. 2014;11:55.

    Not in our library10.1186/1742-2094-11-55

  50. 50.

    Suwanpradid J, Shih M, Pontius L, et al. Arginase1 deficiency in monocytes/macrophages upregulates inducible nitric oxide synthase to promote cutaneous contact hypersensitivity. J Immunol. 2017;199(5):1827‐1834.

    Not in our library10.4049/jimmunol.1700739

  51. 51.

    Wang Y, Inger M, Jiang H, Tenenbaum H, Glogauer M. CD109 plays a role in osteoclastogenesis. PLoS One. 2013;8(4):e61213.

    Not in our library10.1371/journal.pone.0061213

  52. 52.

    Fata JE, Kong YY, Li J, et al. The osteoclast differentiation factor osteoprotegerin‐ligand is essential for mammary gland development. Cell. 2000;103(1):41‐50.

    Not in our library10.1016/s0092-8674(00)00103-3

  53. 53.

    Lin WW, Chen BC, Hsu YW, Lee CM, Shyue SK. Modulation of inducible nitric oxide synthase induction by prostaglandin E2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages. Prostaglandins Other Lipid Mediat. 1999;58(2–4):87‐101.

    Not in our library10.1016/s0090-6980(99)00023-4

  54. 54.

    Thompson CD, Frazier‐Jessen MR, Rawat R, Nordan RP, Brown RT. Evaluation of methods for transient transfection of a murine macrophage cell line, RAW 264.7. Biotechniques. 1999;27(4):pp. 824‐826, 828‐830, 832.

    Not in our library10.2144/99274rr05

  55. 55.

    Nesbitt PD, Lam Y, Thompson LU. Human metabolism of mammalian lignan precursors in raw and processed flaxseed. Am J Clin Nutr. 1999;69(3):549‐555.

    Not in our library10.1093/ajcn/69.3.549

  56. 56.

    Hsu H, Lacey DL, Dunstan CR, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A. 1999;96(7):3540‐3545.

    Not in our library10.1073/pnas.96.7.3540

  57. 57.

    Zauli G, Rimondi E, Nicolin V, Melloni E, Celeghini C, Secchiero P. TNF‐related apoptosis‐inducing ligand (TRAIL) blocks osteoclastic differentiation induced by RANKL plus M‐CSF. Blood. 2004;104(7):2044‐2050.

    Not in our library10.1182/blood-2004-03-1196

  58. 58.

    Narducci P, Nicolin V. Differentiation of activated monocytes into osteoclast‐like cells on a hydroxyapatite substrate: an in vitro study. Ann Anat. 2009;191(4):349‐355.

    Not in our library10.1016/j.aanat.2009.02.009

  59. 59.

    Hotokezaka H, Sakai E, Kanaoka K, et al. U0126 and PD98059, specific inhibitors of MEK, accelerate differentiation of RAW264.7 cells into osteoclast‐like cells. J Biol Chem. 2002;277(49):47366‐47372.

    Not in our library10.1074/jbc.m208284200

  60. 60.

    Shui C, Riggs BL, Khosla S. The immunosuppressant rapamycin, alone or with transforming growth factor‐beta, enhances osteoclast differentiation of RAW264.7 monocyte‐macrophage cells in the presence of RANK‐ligand. Calcif Tissue Int. 2002;71(5):437‐446.

    Not in our library10.1007/s00223-001-1138-3

  61. 61.

    Makihira S, Mine Y, Kosaka E, Nikawa H. Titanium surface roughness accelerates RANKL‐dependent differentiation in the osteoclast precursor cell line, RAW264.7. Dent Mater J. 2007;26(5):739‐745.

    Not in our library10.4012/dmj.26.739

  62. 62.

    Barrett SG, Hansen KS, Bainton DF. Differentiation of cell surface receptors on normal human bone marrow myeloid precursors. Br J Haematol. 1981;48(3):491‐500.

    Not in our library10.1111/j.1365-2141.1981.tb02741.x

  63. 63.

    Dan K, Kuriya S, Nomura T, Hamaguchi H, Sakamaki H. Myeloid and megakaryocytic precursors in the bone marrow and spleen in altered erythropoiesis. Nihon Ketsueki Gakkai Zasshi. 1982;45(1):50‐55.

    Not in our library

  64. 64.

    Charles JF, Hsu LY, Niemi EC, Weiss A, Aliprantis AO, Nakamura MC. Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function. J Clin Invest. 2012;122(12):4592‐4605.

    Not in our library10.1172/jci60920

  65. 65.

    Bord S, Frith E, Ireland DC, Scott MA, Craig JI, Compston JE. Megakaryocytes modulate osteoblast synthesis of type‐l collagen, osteoprotegerin, and RANKL. Bone. 2005;36(5):812‐819.

    Not in our library10.1016/j.bone.2004.12.006

  66. 66.

    Hawa G, Brinskelle‐Schmal N, Glatz K, Maitzen S, Woloszczuk W. Immunoassay for soluble RANKL (receptor activator of NF‐kappaB ligand) in serum. Clin Lab. 2003;49(9–10):461‐463.

    Not in our library

  67. 67.

    Kanamaru F, Iwai H, Ikeda T, Nakajima A, Ishikawa I, Azuma M. Expression of membrane‐bound and soluble receptor activator of NF‐kappaB ligand (RANKL) in human T cells. Immunol Lett. 2004;94(3):239‐246.

    Not in our library10.1016/j.imlet.2004.05.010

  68. 68.

    Melhus H. Soluble RANKL and risk of nontraumatic fracture. JAMA. 2004; 291(22):2703; author reply 2703‐2704.

    Not in our library10.1001/jama.291.22.2703-a

  69. 69.

    Kim DW, Lee HJ, Karmin JA, et al. Mechanical loading differentially regulates membrane‐bound and soluble RANKL availability in MC3T3‐E1 cells. Ann N Y Acad Sci. 2006;1068:568‐572.

    Not in our library10.1196/annals.1346.054

  70. 70.

    Chu EH, Brimer P, Jacobson KB, Merriam EV. Mammalian cell genetics. I. Selection and characterization of mutations auxotrophic for L‐glutamine or resistant to 8‐azaguanine in Chinese hamster cells in vitro. Genetics. 1969;62(2):359‐377.

    Not in our library10.1093/genetics/62.2.359

  71. 71.

    Rhoads JM, Argenzio RA, Chen W, et al. L‐glutamine stimulates intestinal cell proliferation and activates mitogen‐activated protein kinases. Am J Physiol. 1997;272(5 Pt 1):G943‐953.

    Not in our library10.1152/ajpgi.1997.272.5.g943

  72. 72.

    Girven M, Dugdale HF, Owens DJ, Hughes DC, Stewart CE, Sharples AP. l‐glutamine Improves Skeletal Muscle Cell Differentiation and Prevents Myotube Atrophy After Cytokine (TNF‐alpha) Stress Via Reduced p38 MAPK Signal Transduction. J Cell Physiol. 2016;231(12):2720‐2732.

    Not in our library10.1002/jcp.25380

  73. 73.

    Indo Y, Takeshita S, Ishii KA, et al. Metabolic regulation of osteoclast differentiation and function. J Bone Miner Res. 2013;28(11):2392‐2399.

    Not in our library10.1002/jbmr.1976

  74. 74.

    Nguyen J, Nohe A. Factors that Affect the Osteoclastogenesis of RAW264.7 Cells. J Biochem Anal Stud. 2017;2(1).

    Not in our library10.16966/2576-5833.109

  75. 75.

    Schneider M, Marison IW, von Stockar U. The importance of ammonia in mammalian cell culture. J Biotechnol. 1996;46(3):161‐185.

    Not in our library10.1016/0168-1656(95)00196-4

  76. 76.

    Raschke WC, Baird S, Ralph P, Nakoinz I. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell. 1978;15(1):261‐267.

    Not in our library10.1016/0092-8674(78)90101-0

  77. 77.

    Sefton BM, Trowbridge IS, Cooper JA, Scolnick EM. The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid. Cell. 1982;31(2 Pt 1):465‐474.

    Not in our library10.1016/0092-8674(82)90139-8

  78. 78.

    Rosenberg N, Witte ON. The viral and cellular forms of the Abelson (abl) oncogene. Adv Virus Res. 1988;35:39‐81.

    Not in our library10.1016/s0065-3527(08)60708-3

  79. 79.

    Worth LL, Cochran BJ, Haddox MK. Phosphorylation of ornithine decarboxylase at both serine and threonine residues in the ODC‐overproducing, Abelson virus‐transformed RAW264 cell line. Cancer Res. 1994;54(15):3967‐3970.

    Not in our library

  80. 80.

    Fushman D, Xu R, Cowburn D. Direct determination of changes of interdomain orientation on ligation: use of the orientational dependence of 15N NMR relaxation in Abl SH(32). Biochemistry. 1999;38(32):10225‐10230.

    Not in our library10.1021/bi990897g

  81. 81.

    Kim HH, Chung WJ, Lee SW, et al. Association of sustained ERK activity with integrin beta3 induction during receptor activator of nuclear factor kappaB ligand (RANKL)‐directed osteoclast differentiation. Exp Cell Res. 2003;289(2):368‐377.

    Not in our library10.1016/s0014-4827(03)00288-x

  82. 82.

    Izawa T, Zou W, Chappel JC, Ashley JW, Feng X, Teitelbaum SL. c‐Src links a RANK/alphavbeta3 integrin complex to the osteoclast cytoskeleton. Mol Cell Biol. 2012;32(14):2943‐2953.

    Not in our library10.1128/mcb.00077-12

  83. 83.

    Zou W, Croke M, Fukunaga T, Broekelmann TJ, Mecham RP, Teitelbaum SL. Zap70 inhibits Syk‐mediated osteoclast function. J Cell Biochem. 2013;114(8):1871‐1878.

    Not in our library10.1002/jcb.24531

  84. 84.

    Hughes CE, Finney BA, Koentgen F, Lowe KL, Watson SP. The N‐terminal SH2 domain of Syk is required for (hem)ITAM, but not integrin, signaling in mouse platelets. Blood. 2015;125(1):144‐154.

    Not in our library10.1182/blood-2014-05-579375

  85. 85.

    Karst M, Gorny G, Galvin RJ, Oursler MJ. Roles of stromal cell RANKL, OPG, and M‐CSF expression in biphasic TGF‐beta regulation of osteoclast differentiation. J Cell Physiol. 2004;200(1):99‐106.

    Not in our library10.1002/jcp.20036

  86. 86.

    Galibert L, Tometsko ME, Anderson DM, Cosman D, Dougall WC. The involvement of multiple tumor necrosis factor receptor (TNFR)‐associated factors in the signaling mechanisms of receptor activator of NF‐kappaB, a member of the TNFR superfamily. J Biol Chem. 1998;273(51):34120‐34127.

    Not in our library10.1074/jbc.273.51.34120

  87. 87.

    Troen BR. Molecular mechanisms underlying osteoclast formation and activation. Exp Gerontol. 2003;38(6):605‐614.

    Not in our library10.1016/s0531-5565(03)00069-x

  88. 88.

    Darnay BG, Besse A, Poblenz AT, Lamothe B, Jacoby JJ. TRAFs in RANK signaling. Adv Exp Med Biol. 2007;597:152‐159.

    Not in our library10.1007/978-0-387-70630-6_12

  89. 89.

    Darnay BG, Haridas V, Ni J, Moore PA, Aggarwal BB. Characterization of the intracellular domain of receptor activator of NF‐kappaB (RANK). Interaction with tumor necrosis factor receptor‐associated factors and activation of NF‐kappab and c‐Jun N‐terminal kinase. J Biol Chem. 1998;273(32):20551‐20555.

    Not in our library10.1074/jbc.273.32.20551

  90. 90.

    Gratacap MP, Guillermet‐Guibert J, Martin V, et al. Regulation and roles of PI3Kbeta, a major actor in platelet signaling and functions. Adv Enzyme Regul. 2011;51(1):106‐116.

    Not in our library10.1016/j.advenzreg.2010.09.011

  91. 91.

    Ha H, Kwak HB, Lee SW, et al. Reactive oxygen species mediate RANK signaling in osteoclasts. Exp Cell Res. 2004;301(2):119‐127.

    Not in our library10.1016/j.yexcr.2004.07.035

  92. 92.

    Sun L, Peng Y, Zaidi N, et al. Evidence that calcineurin is required for the genesis of bone‐resorbing osteoclasts. Am J Physiol Renal Physiol. 2007;292(1):F285‐291.

    Not in our library10.1152/ajprenal.00415.2005

  93. 93.

    Baek JM, Park SH, Cheon YH, et al. Esculetin attenuates receptor activator of nuclear factor kappa‐B ligand‐mediated osteoclast differentiation through c‐Fos/nuclear factor of activated T‐cells c1 signaling pathway. Biochem Biophys Res Commun. 2015;461(2):334‐341.

    Not in our library10.1016/j.bbrc.2015.04.034

  94. 94.

    Kim WS, Kim HJ, Lee ZH, Lee Y, Kim HH. Apolipoprotein E inhibits osteoclast differentiation via regulation of c‐Fos, NFATc1 and NF‐kappaB. Exp Cell Res. 2013;319(4):436‐446.

    Not in our library10.1016/j.yexcr.2012.12.004

  95. 95.

    Zhu LL, Zaidi S, Moonga BS, Troen BR, Sun L. RANK‐L induces the expression of NFATc1, but not of NFkappaB subunits during osteoclast formation. Biochem Biophys Res Commun. 2005;326(1):131‐135.

    Not in our library10.1016/j.bbrc.2004.10.212

  96. 96.

    Yip KH, Feng H, Pavlos NJ, Zheng MH, Xu J. p62 Ubiquitin binding‐associated domain mediated the receptor activator of nuclear factor‐kappaB ligand‐induced osteoclast formation: a new insight into the pathogenesis of Paget's disease of bone. Am J Pathol. 2006;169(2):503‐514.

    Not in our library10.2353/ajpath.2006.050960

  97. 97.

    Hasegawa H, Kido S, Tomomura M, et al. Serum calcium‐decreasing factor, caldecrin, inhibits osteoclast differentiation by suppression of NFATc1 activity. J Biol Chem. 2010;285(33):25448‐25457.

    Not in our library10.1074/jbc.m109.068742

  98. 98.

    Zhang Y, Jiang P, Li W, et al. Calcineurin/NFAT signaling pathway mediates titanium particleinduced inflammation and osteoclast formation by inhibiting RANKL and MCSF in vitro. Mol Med Rep. 2017;16(6):8223‐8230.

    Not in our library10.3892/mmr.2017.7670

  99. 99.

    Ye F, Zhou Q, Tian L, Lei F, Feng D. The protective effect of berberine hydrochloride on LPSinduced osteoclastogenesis through inhibiting TRAF6Ca2+calcineurinNFATcl signaling pathway. Mol Med Rep. 2017;16(5):6228‐6233.

    Not in our library10.3892/mmr.2017.7338

  100. 100.

    Jeong JC, Lee JW, Yoon CH, Kim HM, Kim CH. Drynariae Rhizoma promotes osteoblast differentiation and mineralization in MC3T3‐E1 cells through regulation of bone morphogenetic protein‐2, alkaline phosphatase, type I collagen and collagenase‐1. Toxicol In Vitro. 2004;18(6):829‐834.

    Not in our library10.1016/j.tiv.2004.05.002

  101. 101.

    Kim MH, Kim BT, Min YK, Kim SH. Profiling signalling pathways of the receptor activator of NF‐kappaB ligand‐induced osteoclast formation in mouse monocyte cells, RAW264.7. Amino Acids. 2008;34(3):497‐506.

    Not in our library10.1007/s00726-006-0461-4

  102. 102.

    Sismey‐Durrant HJ, Atkinson SJ, Hopps RM, Heath JK. The effect of lipopolysaccharide from bacteroides gingivalis and muramyl dipeptide on osteoblast collagenase release. Calcif Tissue Int. 1989;44(5):361‐363.

    Not in our library10.1007/bf02556318

  103. 103.

    Kikkawa I, Saito S, Tominaga K, Hoshino Y, Ooi Y, Nakano M. Lipopolysaccharide (LPS) stimulates the production of tumor necrosis factor (TNF)‐alpha and expression of inducible nitric oxide synthase (iNOS) by osteoclasts (OCL) in murine bone marrow cell culture. Microbiol Immunol. 1998;42(9):591‐598.

    Not in our library10.1111/j.1348-0421.1998.tb02329.x

  104. 104.

    De Santa F, Vitiello L, Torcinaro A, Ferraro E. The role of metabolic remodeling in macrophage polarization and its effect on skeletal muscle regeneration. Antioxid Redox Signal. 2018.

    Not in our library10.1089/ars.2017.7420

  105. 105.
    Molecular Mechanisms That Influence the Macrophage M1–M2 Polarization Balance · 2014
    No notice
  106. 106.

    Yang J, Zhu Y, Duan D, et al. Enhanced activity of macrophage M1/M2 phenotypes in periodontitis. Arch Oral Biol. 2018;96:234‐242.

    Not in our library10.1016/j.archoralbio.2017.03.006

  107. 107.

    Mizutani H, Ishihara Y, Izawa A, et al. Lipopolysaccharide of Aggregatibacter actinomycetemcomitans up‐regulates inflammatory cytokines, prostaglandin E2 synthesis and osteoclast formation in interleukin‐1 receptor antagonist‐deficient mice. J Periodontal Res. 2013;48(6):748‐756.

    Not in our library10.1111/jre.12065

  108. 108.

    Scianaro R, Insalaco A, Bracci Laudiero L, et al. Deregulation of the IL‐1beta axis in chronic recurrent multifocal osteomyelitis. Pediatr Rheumatol Online J. 2014;12:30.

    Not in our library10.1186/1546-0096-12-30

  109. 109.

    Sells Galvin RJ, Gatlin CL, Horn JW, Fuson TR. TGF‐beta enhances osteoclast differentiation in hematopoietic cell cultures stimulated with RANKL and M‐CSF. Biochem Biophys Res Commun. 1999;265(1):233‐239.

    Not in our library10.1006/bbrc.1999.1632

  110. 110.

    Islam S, Hassan F, Tumurkhuu G, et al. Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells. Biochem Biophys Res Commun. 2007;360(2):346‐351.

    Not in our library10.1016/j.bbrc.2007.06.023

  111. 111.

    Hotokezaka H, Sakai E, Ohara N, et al. Molecular analysis of RANKL‐independent cell fusion of osteoclast‐like cells induced by TNF‐alpha, lipopolysaccharide, or peptidoglycan. J Cell Biochem. 2007;101(1):122‐134.

    Not in our library10.1002/jcb.21167

  112. 112.

    Siddiqi MH, Siddiqi MZ, Kang S, et al. Inhibition of osteoclast differentiation by ginsenoside Rg3 in RAW264.7 cells via RANKL, JNK and p38 MAPK pathways through a modulation of cathepsin K: an in silico and in vitro study. Phytother Res. 2015;29(9):1286‐1294.

    Not in our library10.1002/ptr.5374

  113. 113.

    Nakanishi‐Matsui M, Yano S, Matsumoto N, Futai M. Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity. Biochem Biophys Res Commun. 2012;425(2):144‐149.

    Not in our library10.1016/j.bbrc.2012.07.050

  114. 114.

    Piper K, Boyde A, Jones SJ. The relationship between the number of nuclei of an osteoclast and its resorptive capability in vitro. Anat Embryol (Berl). 1992;186(4):291‐299.

    Not in our library10.1007/bf00185977

  115. 115.

    Lees RL, Heersche JN. Macrophage colony stimulating factor increases bone resorption in dispersed osteoclast cultures by increasing osteoclast size. J Bone Miner Res. 1999;14(6):937‐945.

    Not in our library10.1359/jbmr.1999.14.6.937

  116. 116.

    Lees RL, Heersche JN. Differences in regulation of pH(i) in large (>/=10 nuclei) and small (</=5 nuclei) osteoclasts. Am J Physiol Cell Physiol. 2000;279(3):C751‐761.

    Not in our library10.1152/ajpcell.2000.279.3.c751

  117. 117.

    Manolson MF, Yu H, Chen W, et al. The a3 isoform of the 100‐kDa V‐ATPase subunit is highly but differentially expressed in large (>or=10 nuclei) and small (<or= nuclei) osteoclasts. J Biol Chem. 2003;278(49):49271‐49278.

    Not in our library10.1074/jbc.m309914200

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    Nordstrom T, Shrode LD, Rotstein OD, et al. Chronic extracellular acidosis induces plasmalemmal vacuolar type H+ ATPase activity in osteoclasts. J Biol Chem. 1997;272(10):6354‐6360.

    Not in our library10.1074/jbc.272.10.6354

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    Zou W, Bar‐Shavit Z. Dual modulation of osteoclast differentiation by lipopolysaccharide. J Bone Miner Res. 2002;17(7):1211‐1218.

    Not in our library10.1359/jbmr.2002.17.7.1211

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    Suda K, Woo JT, Takami M, Sexton PM, Nagai K. Lipopolysaccharide supports survival and fusion of preosteoclasts independent of TNF‐alpha, IL‐1, and RANKL. J Cell Physiol. 2002;190(1):101‐108.

    Not in our library10.1002/jcp.10041

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    Shuto T, Jimi E, Kukita T, Hirata M, Koga T. Granulocyte‐macrophage colony stimulating factor suppresses lipopolysaccharide‐induced osteoclast‐like cell formation in mouse bone marrow cultures. Endocrinology. 1994;134(2):831‐837.

    Not in our library10.1210/endo.134.2.8299579

  122. 122.

    Barbour SE, Wong C, Rabah D, Kapur A, Carter AD. Mature macrophage cell lines exhibit variable responses to LPS. Mol Immunol. 1998;35(14–15):977‐987.

    Not in our library10.1016/s0161-5890(98)00070-4

  123. 123.

    Shiina Y, Yamaguchi A, Yamana H, Abe E, Yoshiki S, Suda T. Comparison of the mechanisms of bone resorption induced by 1 alpha,25‐dihydroxyvitamin D3 and lipopolysaccharides. Calcif Tissue Int. 1986;39(1):28‐34.

    Not in our library10.1007/bf02555737

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    Yang S, Takahashi N, Yamashita T, et al. Muramyl dipeptide enhances osteoclast formation induced by lipopolysaccharide, IL‐1 alpha, and TNF‐alpha through nucleotide‐binding oligomerization domain 2‐mediated signaling in osteoblasts. J Immunol. 2005;175(3):1956‐1964.

    Not in our library10.4049/jimmunol.175.3.1956

  125. 125.

    Kikuchi T, Matsuguchi T, Tsuboi N, et al. Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via Toll‐like receptors. J Immunol. 2001;166(5):3574‐3579.

    Not in our library10.4049/jimmunol.166.5.3574

  126. 126.

    Nair SP, Meghji S, Wilson M, Reddi K, White P, Henderson B. Bacterially induced bone destruction: mechanisms and misconceptions. Infect Immun. 1996;64(7):2371‐2380.

    Not in our library10.1128/iai.64.7.2371-2380.1996

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    Sakthiswary R, Das S. The effects of TNF alpha antagonist therapy on bone metabolism in rheumatoid arthritis: a systematic review. Curr Drug Targets. 2013;14(13):1552‐1557.

    Not in our library10.2174/13894501113149990176

  128. 128.

    Boyce BF, Li P, Yao Z, et al. TNF‐alpha and pathologic bone resorption. Keio J Med. 2005;54(3):127‐131.

    Not in our library10.2302/kjm.54.127

  129. 129.

    Zha L, He L, Liang Y, et al. TNF‐alpha contributes to postmenopausal osteoporosis by synergistically promoting RANKL‐induced osteoclast formation. Biomed Pharmacother. 2018;102:369‐374.

    Not in our library10.1016/j.biopha.2018.03.080

  130. 130.

    Kobayashi K, Takahashi N, Jimi E, et al. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL‐RANK interaction. J Exp Med. 2000;191(2):275‐286.

    Not in our library10.1084/jem.191.2.275

  131. 131.

    Quinn JM, Itoh K, Udagawa N, et al. Transforming growth factor beta affects osteoclast differentiation via direct and indirect actions. J Bone Miner Res. 2001;16(10):1787‐1794.

    Not in our library10.1359/jbmr.2001.16.10.1787

  132. 132.

    Li P, Schwarz EM, O'Keefe RJ, Ma L, Boyce BF, Xing L. RANK signaling is not required for TNFalpha‐mediated increase in CD11(hi) osteoclast precursors but is essential for mature osteoclast formation in TNFalpha‐mediated inflammatory arthritis. J Bone Miner Res. 2004;19(2):207‐213.

    Not in our library10.1359/jbmr.0301233

  133. 133.

    Blair HC, Athanasou NA. Recent advances in osteoclast biology and pathological bone resorption. Histol Histopathol. 2004;19(1):189‐199.

    Not in our library10.14670/hh-19.189

  134. 134.

    Nakao A, Fukushima H, Kajiya H, Ozeki S, Okabe K. RANKL‐stimulated TNFalpha production in osteoclast precursor cells promotes osteoclastogenesis by modulating RANK signaling pathways. Biochem Biophys Res Commun. 2007;357(4):945‐950.

    Not in our library10.1016/j.bbrc.2007.04.058

  135. 135.

    Nakao J, Fujii Y, Kusuyama J, et al. Low‐intensity pulsed ultrasound (LIPUS) inhibits LPS‐induced inflammatory responses of osteoblasts through TLR4‐MyD88 dissociation. Bone. 2014;58:17‐25.

    Not in our library10.1016/j.bone.2013.09.018

  136. 136.

    Zou W, Amcheslavsky A, Takeshita S, Drissi H, Bar‐Shavit Z. TNF‐alpha expression is transcriptionally regulated by RANK ligand. J Cell Physiol. 2005;202(2):371‐378.

    Not in our library10.1002/jcp.20127

  137. 137.

    Qi MY, Wang ZZ, Zhang Z, et al. AU‐rich‐element‐dependent translation repression requires the cooperation of tristetraprolin and RCK/P54. Mol Cell Biol. 2012;32(5):913‐928.

    Not in our library10.1128/mcb.05340-11

  138. 138.

    Park JW, Jeon YJ, Lee JC, et al. Destabilization of TNF‐alpha mRNA by Rapamycin. Biomol Ther (Seoul). 2012;20(1):43‐49.

    Not in our library10.4062/biomolther.2012.20.1.043

  139. 139.

    Amirouche A, Tadesse H, Lunde JA, Belanger G, Cote J, Jasmin BJ. Activation of p38 signaling increases utrophin A expression in skeletal muscle via the RNA‐binding protein KSRP and inhibition of AU‐rich element‐mediated mRNA decay: implications for novel DMD therapeutics. Hum Mol Genet. 2013;22(15):3093‐3111.

    Not in our library10.1093/hmg/ddt165

  140. 140.

    Degese MS, Tanos T, Naipauer J, et al. An interplay between the p38 MAPK pathway and AUBPs regulates c‐fos mRNA stability during mitogenic stimulation. Biochem J. 2015;467(1):77‐90.

    Not in our library10.1042/bj20141100

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    Shinohara H, Teramachi J, Okamura H, Yang D, Nagata T, Haneji T. Double stranded RNA‐dependent protein kinase is necessary for tnf‐alpha‐induced osteoclast formation in vitro and in vivo. J Cell Biochem. 2015;116(9):1957‐1967.

    Not in our library10.1002/jcb.25151

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