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

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.


| INTRODUC TI ON
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 splenocytes 5 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  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 functions 21 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.

| PHENOT YPE S TUDY OF R AW26 4.7 AND ITS ROLE ON THE REG UL ATI ON OF R AW-O C S
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 al 4 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 al 51 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 formation 51 ( Figure 1).

| R ANKL AND ITS ROLE IN R AW-O C S INDUC TION
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 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. F I G U R E 2 Schematic of RANKL, LPS and TNF-a signalling for RAW-OCs 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

| LIP OP OLYSACCHARIDE AND ITS ROLE IN R AW-O C S INDUC TI ON
Physiological defense responding is important for the host to survive infection. 76 104 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. 107 113 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 LPSinduced 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

| TNF-α AND ITS ROLE IN R AW-O C S INDUC TION
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 arthritis 127 and aseptic periprosthetic osteolysis. 128  association is necessary for TNF-a induced RAW-OCs formation by p38MAPK, and ERK signalling (Figure 2).

E THI C S APPROVAL AND CON S ENT TO PARTI CIPATE
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'.

CON S ENT FOR PUB LI C ATI ON
The manuscript is approved by all authors for publication.

AVAIL AB ILIT Y OF DATA AND MATERIAL S
All data and materials were included in the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.