Research Area

Osteoclast


Introduction of Osteoclast

Osteoclast

Currently, in vitro culture of osteoclasts is based on the theory that "osteocytes are a fusion of hematopoietic monocyte precursor cells." That is, mononuclear cells are isolated from bone marrow or peripheral blood and spleen, and some cytokines, hormones and other inducers are added to induce monocyte differentiation, and finally, multinucleated osteoclasts are formed. Whether or not osteoclasts originate from hematopoietic stem cells is still questionable. Therefore, the origin of osteoclasts is still a hot topic of current research. It is important to understand their expression of special marker and surface-specific antigens during the differentiation and formation of osteoclasts. Studying these mechanisms can help in the treatment of related diseases.

Expression Makers of Osteoclast

Now introduce some markers that can be specifically used to label osteoclasts: Marker enzyme TRAP: Immunohistochemical studies have detected that osteoclasts contain high levels of acid hydrolase. Tartrate-resistant acid phosphatase (TLAP), also known as purple acid phosphatase, which is highly expressed in osteoclasts, is present in extracellular channels of ribosomes, Golgi folds, and the gap between cells and bone. It has been thought in the past that the distribution of TRAP is mainly restricted to osteoclasts of normal individuals, hairy cell leukemia cells or spleen macrophages of patients with Gaucher disease. However, recent studies using PR-PCR technology have shown that TRAP is expressed in many tissues, including the intestines, kidneys, and lungs. In normal tissues, bone has the highest expression level of TRAP, and TRAP can even act as a marker enzyme for osteoclasts. Carbonic anhydrase II: The reverse construction of carbonic anhydrase II inhibits the bone-absorbing of osteoclasts, whether in isolated osteoclasts or in tissue culture of bone. Asotra and his colleagues found in rabbits that osteoclasts that are actively absorbed exhibit higher levels of carbonic anhydrase II mRNA than other osteoclasts. Osteoclast-like cells isolated from human giant cell tumors also have this feature. Cathepsin: Recently, a novel human cysteine protease named cathepsin K or cathepsin O has been cloned, an enzyme cloned by Inaoka and colleagues from a rabbit osteoclast cDNA library. Very similar, the high-level expression of cathepsin K in osteoblasts of the osteoarthritis and giant cells of giant cell tumor of the bone suggests that it plays a role in bone resorption. MMP9: Another marker for osteoclasts is MMP-9, also known as 92 kDa gelatinase or type IV collagenase. Many studies have shown that MMP-9 is only found in normal individuals and osteoclasts in the bone tissues of patients with rheumatoid arthritis and metastatic cancer. Okada and colleagues found that MMP-9 is produced by osteoclasts in human bone tissue, which together with MMP-1 and cysteine proteases degrade collagen under the pleats. Wucherpfenning et al. reported that high levels of MMP 9 are present in giant cells in human osteoblasts and osteoclasts in patients with Paget's disease. Their differentiation of human osteoclast cDNA library revealed a large amount of MMP-9 expression, suggesting that it plays an important role in the degradation of collagen in the bone resorption compartment during bone remodeling. Non-specific surface phenotype proteins: Athanasou and colleagues describe the surface phenotype of osteoclasts in detail. They found that osteoclasts from humans, rabbits, and birds can react with a monoclonal antibody that targets the human β1-integrin and macrophage CD68-associated antigen. In addition, the osteoclasts of the birds can also react with CD11a/18 and CD14, which are not present in human osteoclasts. Osteoclasts have no Fc receptor compared to multinucleated macrophages. The antigen of the Kn22 antibody is a 50 kDa protein that is expressed on the surface of osteoclasts, but its properties and functions are unknown. The best distinguishing factor for calcitonin receptors is the calcitonin receptor. Calcitonin receptors are expressed in many cells, including kidney and brain cells, but are not expressed in multinucleated macrophages. Recently, several organisms have been cloned for calcitonin receptors, and at least two isoforms have been identified. Ikegami and colleagues have reported that one of the isoforms called C1A is clearly expressed in osteoclasts; Goldring et al. also detected calcitonin receptor isoforms in giant cells in human osteoclasts expression. Takahashi et al. pointed out that the expression of calcitonin receptor appeared later in the process of osteoclast differentiation, usually after the mother cell has been shaped into the osteoclast lineage, that is, after the mitosis stage. PTH receptors have historically considered that osteoclasts do not contain PTH receptors, but Agarwala and Gay, as well as Teti, report that osteoclasts in rodents and birds have PTH receptors. However, the importance of PTH receptors in mature osteoclasts is unknown because high-purity mature osteoclasts do not respond to PTH. Hakeda et al. used immunocytochemistry to discover that osteoclasts derived from murine hematopoietic progenitor cells contain parathyroid receptors; this suggests that PTH may act directly on osteoclasts to induce their differentiation. Similar reports have been reported by Kurihara et al.: PTH is a highly purified mitogen of human osteoclasts, suggesting that PTH has a direct effect on osteoclasts. Therefore, it is likely that osteoclasts respond directly to PTH, but mature osteoclasts, although they also contain PTH receptors, do not directly respond to PTH. Pp60csrc: Studies have also shown that osteoclasts also have high levels of expression of pp60csrc, a receptor-free tyrosine kinase. The expression levels of pp60c src protein and kinase in osteoclasts are almost as high as in brain and platelets. This contrasts with other bone cells because the expression level of the pp60c-src protein is very low in other bone cells. Moreover, when 1, 2 5 -dihydroxy vitamin D3 induces bone marrow cells to express a certain osteoclast phenotype, high levels of pp60csrc protein are often present in these cells. Osteoclasts also express three other src-like kinases; they are c-Fym, c-Yes and c-Lyn, but these proteins do not play a role in the osteoclastic absorption of bone; because of inhibition of pp60 in osteoclasts, the activity of c-src inhibits bone absorption. Soriano and colleagues used homologous recombination techniques to find severe osteopetrosis in transgenic mice lacking the pp60c-src gene. Tanaka and colleagues further found that high-level expression of pp60 csrc by osteoclasts first appeared on the pleated side. Therefore, although pp60csrc is expressed in a variety of cells, its high level of expression is one of the characteristics of osteoclasts, and src tyrosine kinase activity plays an important role in bone resorption.

Research Status of Osteoclast

Osteoclasts (OC) are differentiated from proto-mononuclear cells in bone marrow hematopoietic stem cells and are mainly found in the periosteum. OC is the only cell with bone resorption that acts primarily for bone resorption, initiates bone remodeling and clears old bone matrix. When the function of OC is over-expressed, it will cause excessive absorption of bone matrix, causing osteolytic diseases such as osteoporosis. By studying the OC differentiation and maturation pathway, it can provide new guidance for the treatment of osteolytic diseases. Differentiation of OC can be divided into four stages: bone marrow-derived: primary monocytes differentiate into specific hematopoietic progenitors; hematopoietic progenitors: forward osteoclast differentiation; OCP further differentiates into tartrate-resistant acid phosphatase-positive pre-osteocytes (OCP); OCP finally gathers and assembles into mature OCs. The major OC-forming pathways are currently found to be nuclear factor receptor activating factor ligand-nuclear factor receptor activating factor-osteoprotegerin (RANKL-RANK-OPG) signaling pathway, macrophage colony-stimulating factor (M- CSF) signaling pathway and immunoreceptor tyrosine activation motif (ITAM) signaling pathway. The RANKL-RANK-OPG signaling pathway is currently the most important OC differentiation pathway have been found. The RANKL-RANK-OPG signal pathway consists of three factors: RANK, RANKL, and OPG. The RANK expression can be detected on the surface of OC and is a member of the tumor necrosis factor receptor (TNFR) superfamily. RANKL, also known as OC differentiation factor, is a type II transmembrane protein that can induce differentiation and maturation of OC under the stimulation of M-CSF. It belongs to the tumor necrosis factor (TNF) family and can be found in mature OC or thymocytes. Experiments have shown that under certain conditions, interleukin (IL)-1, IL-6, IL-8, TNF-α, and other factors can induce OC differentiation under RANKL-free conditions. These factors are also known as RANKL replacement factors. Upon receiving the signal of RANKL, RANK on the OC surface immediately initiates differentiation and formation of OC and simultaneously inhibits apoptosis of OC. OPG is another receptor for RANKL, also known as osteoclastogenesis inhibitory factor (OCIF), which competes with RANKL for binding to RANK and inhibits OC differentiation. Nuclear factor κB (NF-κB) can be divided into typical and atypical pathways in the RANKL-RANK-OPG signaling pathway. In the classical cell pathway, RANK, by binding to RANKL, can cause phosphorylation of nuclear factor-kappaB inhibitor kinase β (IKKβ), resulting in the degradation of nuclear factor-kappa B inhibitory protein (IKB), allowing NF-κB to enter the nucleus and regulate OC. The differentiation also prevented the apoptosis of OC. The RANKL-RANK-OPG signaling pathway triggers the activation of the receptor Tyr kinase and recruit’s growth factor receptor binding protein 2 (Grb2), and Grb2 forms a complex with SOS, which in turn activates mitogen-activated protein kinase. The kinase, mitogen-activated protein kinase, and mitogen-activated protein kinase ultimately activate extracellular signal-regulated kinase. The activated ERK enters the nucleus, and the transcription factor ELK is activated by ERK and the sequence of the c-fos gene promoter regulates the combination of the serum response element and ERK to promote OC differentiation.

References:

  1. Dai Q, Xie F, Han Y, et al. Inactivation of Regulatory-associated Protein of mTOR (Raptor)/Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Osteoclasts Increases Bone Mass by Inhibiting Osteoclast Differentiation in Mice. Journal of Biological Chemistry. 2017, 292(1):196-204.
  2. Kim M H, Ryu SYChoi J S. Saurolactam inhibits osteoclast differentiation and stimulates apoptosis of mature osteoclasts. Journal of Cellular Physiology. 2010, 221(3):618-628.
  3. Lee D E, Kim J H, Choi S H, et al. Periodontitis mainly increases osteoclast formation via enhancing the differentiation of quiescent osteoclast precursors into osteoclasts. Journal of Periodontal Research. 2015, 50(2):256-264.
  4. Aassk D, Algate K, Coleman R, et al. Osteoclast-Associated Receptor (OSCAR) Distribution in the Synovial Tissues of Patients with Active RA and TNF-α and RANKL Regulation of Expression by Osteoclasts In Vitro. Inflammation. 2017, 40(5):1566-1575.
  5. Fujihara Y, Kondo H, Noguchi T, et al. Glucocorticoids mediate circadian timing in peripheral osteoclasts resulting in the circadian expression rhythm of osteoclast-related gene. Bone. 2014, 61(4):1-9.

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