Properties of RANKL/RANK in Differentiation and Metastasis
Properties of RANKL/RANK in Differentiation and Metastasis
Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage. Differentiation of osteoclasts is dependent on stromal cells such as osteoblasts; however, few molecular details emerged until OPG was identified. Two different groups discovered that OPG inhibited bone destruction and osteoclastogenesis. OPG belongs to the TNF receptor family but lacks a transmembrane domain. RANKL, which was previously reported as a dendritic cell survival factor, was identified as a ligand for OPG. Addition of RANKL to bone marrow culture induces osteoclast differentiation in the presence of M-CSF.RANKL and RANK knockout mice displayed osteopetrosis owing to a defect in osteoclastogenesis, revealing that RANKL signals are essential for osteoclastogenesis in vivo. RANKL also stimulates multinucleation from osteoclast precursor cells and bone resorption activity of mature osteoclasts. The survival of mature osteoclasts is regulated by several factors including RANKL. Thus, RANKL is involved in the differentiation, activation and survival of osteoclasts (Figure 1). In normal physiology, RANKL controls bone homeostasis by co-operating with OPG.
(Enlarge Image)
Figure 1.
Role of RANKL in differentiation, fusion, activation and survival of osteoclasts. Osteoclast differentiation is a multistep process. Myeloid progenitor cells progressively differentiate into osteoclast precursor cells, mononuclear preosteoclasts and finally multinucleated osteoclasts. Osteoclasts are further activated to become mature bone-resorbing cells. RANKL produced from osteoblasts and osteocytes controls all of these processes. After osteoclasts finish bone resorption they undergo apoptosis, which is also regulated by RANKL.
RANKL expression is induced by stimulating osteoblasts with several osteotropic factors such as parathyroid hormone (PTH), vitamin D3 and IL-6, which enhance osteoclastogenesis. Membrane-bound RANKL expressed on the surface of osteoblasts has been thought to be important for osteoclastogenesis. However, two recent studies have provided evidence that high expression of RANKL in osteocytes plays a crucial role in osteoclastogenesis, especially in bone remodeling. Osteocytes are embedded cells in bone matrix and are terminally differentiated cells derived from osteoblasts. They communicate with each other through long cytoplasmic projections that form a network, enabling nutrient and waste transport and response to mechanical stimuli. Osteocytes secrete soluble factors such as sclerostin, which controls bone formation. It has been suggested that soluble and/or membrane forms of RANKL derived from osteocytes are involved in osteoclastogenesis. RANKL is also expressed in lymphocytes. RANKL expressed by T- or B-lymphocytes does not play an important role in bone remodeling under normal physiological conditions; however, RANKL expressed in B cells was partially involved in a mouse model of bone loss induced by estrogen deficiency. In osteoclast precursor cells, the expression of RANK is also stimulated by several factors including M-CSF, TNF-α, LPS and WNT5A.
Engagement of RANKL with RANK expressed on osteoclast precursor cells induces intracellular signals essential for osteoclast differentiation and activation (Figure 1). Signaling through RANK is mediated by recruitment of TRAFs. Among the TRAF family of proteins, TRAF6 is essential for RANKL-induced osteoclastogenesis. TRAF6 activates downstream signaling components such as NF-κB and MAPK. RANKL promotes osteoclast differentiation via activation of the NF-κB pathway. RANK signaling also activates the transcription factor AP-1 by inducing its component c-Fos. Activation of NF-κB and c-Fos leads to the induction of NFATc1, which stimulates the expression of osteoclast-specific genes such as cathepsin K. RANKL also induces the phosphorylation of MITF, which is another transcription factor involved in osteoclastogenesis that is activated downstream of MAPK. The transcription factor complex, including transcription factors such as NFATc1 and MITF, stimulates the expression of osteoclast-specific genes such as cathepsin K. In osteoclastogenesis, the expression of NFATc1 is strongly induced via autoamplification in the later stages of differentiation. Transcriptional activity of NFATc1 is dependent on the localization of protein in nuclei, which is regulated by calcium signaling. Costimulatory molecules possessing tyrosine-based activation motif-harboring adaptors induce calcium signaling in osteoclastogenesis. In addition, RANK has a unique cytoplasmic domain that recruits adaptor proteins Gab2 and PLCγ2, which also induce calcium signaling. Phosphorylation of PLCγ2 in conjunction with costimulatory signals lead to the activation of NFATc1. NFATc1 also stimulates transcription of the D2 isoform of the vacuolar ATPase Vo domain and of the dendritic cell-specific transmembrane protein DC-STAMP, which are involved in multinucleation of osteoclasts. Thus, it is generally accepted that NFATc1 is involved in the differentiation and multinucleation of osteoclasts.
During bone resorption, osteoclasts attach to the bone surface, acidify and solubilize the mineral portion of the matrix, and degrade the organic matrix of bone by proteinases such as cathepsin K in the sealed bone–cell interface. Recently, another transcription factor, LRF (also previously known as OCZF in rats and FBI-1 in humans), was shown to be involved in bone resorption activity of osteoclasts. LRF (also called POKEMON), previously reported to be proto-oncogene, is a regulator of differentiation of hematopoietic cells including B and T cells, and erythrocytes. OCZF is expressed in mature osteoclasts in vivo, and the expression is induced by RANKL in osteoclastogenesis in vitro. LRF together with NFATc1 increases the transcriptional activity of cathepsin K, thereby increasing bone resorption. Activation of bone resorption involves polarization of osteoclasts. Polarization requires activation of c-Src by integrin αvβ3, which induces reorganization of the cytoskeleton. It was recently shown that RANKL directly affects this process. Treatment of prefusion osteoclasts with RANKL induces c-Src to associate with RANK and cytoskeletal-organizing molecules such as Syk, Vav3 and Rac1. In osteoclasts, Rho GTPases such as RhoA, Rac1 and Cdc42 control the organization of F-actin, and the vesicular trafficking pathway is regulated by Rab7, which is a GTPase member of the Rab family.
Integrin-mediated signaling in osteoclasts, which is also important for the survival of osteoclasts, is regulated by several cytokines including RANKL. Downstream of RANK, c-Src activates PI3K and AKT, which subsequently phosphorylates BAD and inactivates caspase 9, thereby preventing activation of the apoptosis cascade. JNK and NF-κB activated by RANKL increase the expression of antiapoptosis genes, such as Bcl-2 and Bcl-xl. RANKL also reduces the level of Fas expression in mature osteoclasts, thereby reducing Fas-mediated apoptosis. Osteoclasts express TRAIL, which is involved in osteoclast apoptosis. OPG is a decoy receptor for RANKL, but also binds and inhibits TRAIL. By inhibiting TRAIL, OPG directly decreases apoptosis of osteoclasts.
The action of RANKL/RANK/OPG in osteoclastogenesis is influenced by the microenvironment. Extracellular matrix components, such as glycosaminoglycans (GAGs) including hyaluronan and heparin, affect osteoclastogenesis. Heparin and GAGs inhibit adherence and spreading of osteoclast precursors and inhibit osteoclast differentiation. By contrast, heparin enhances bone resorption of osteoclasts by inhibiting OPG function in the C-terminal heparin binding domain. It has been reported that GAGs highly expressed in osteosarcoma may regulate RANKL function by inhibiting RANKL–OPG interactions.
Role of RANKL in Osteoclastogenesis
Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage. Differentiation of osteoclasts is dependent on stromal cells such as osteoblasts; however, few molecular details emerged until OPG was identified. Two different groups discovered that OPG inhibited bone destruction and osteoclastogenesis. OPG belongs to the TNF receptor family but lacks a transmembrane domain. RANKL, which was previously reported as a dendritic cell survival factor, was identified as a ligand for OPG. Addition of RANKL to bone marrow culture induces osteoclast differentiation in the presence of M-CSF.RANKL and RANK knockout mice displayed osteopetrosis owing to a defect in osteoclastogenesis, revealing that RANKL signals are essential for osteoclastogenesis in vivo. RANKL also stimulates multinucleation from osteoclast precursor cells and bone resorption activity of mature osteoclasts. The survival of mature osteoclasts is regulated by several factors including RANKL. Thus, RANKL is involved in the differentiation, activation and survival of osteoclasts (Figure 1). In normal physiology, RANKL controls bone homeostasis by co-operating with OPG.
(Enlarge Image)
Figure 1.
Role of RANKL in differentiation, fusion, activation and survival of osteoclasts. Osteoclast differentiation is a multistep process. Myeloid progenitor cells progressively differentiate into osteoclast precursor cells, mononuclear preosteoclasts and finally multinucleated osteoclasts. Osteoclasts are further activated to become mature bone-resorbing cells. RANKL produced from osteoblasts and osteocytes controls all of these processes. After osteoclasts finish bone resorption they undergo apoptosis, which is also regulated by RANKL.
RANKL expression is induced by stimulating osteoblasts with several osteotropic factors such as parathyroid hormone (PTH), vitamin D3 and IL-6, which enhance osteoclastogenesis. Membrane-bound RANKL expressed on the surface of osteoblasts has been thought to be important for osteoclastogenesis. However, two recent studies have provided evidence that high expression of RANKL in osteocytes plays a crucial role in osteoclastogenesis, especially in bone remodeling. Osteocytes are embedded cells in bone matrix and are terminally differentiated cells derived from osteoblasts. They communicate with each other through long cytoplasmic projections that form a network, enabling nutrient and waste transport and response to mechanical stimuli. Osteocytes secrete soluble factors such as sclerostin, which controls bone formation. It has been suggested that soluble and/or membrane forms of RANKL derived from osteocytes are involved in osteoclastogenesis. RANKL is also expressed in lymphocytes. RANKL expressed by T- or B-lymphocytes does not play an important role in bone remodeling under normal physiological conditions; however, RANKL expressed in B cells was partially involved in a mouse model of bone loss induced by estrogen deficiency. In osteoclast precursor cells, the expression of RANK is also stimulated by several factors including M-CSF, TNF-α, LPS and WNT5A.
Engagement of RANKL with RANK expressed on osteoclast precursor cells induces intracellular signals essential for osteoclast differentiation and activation (Figure 1). Signaling through RANK is mediated by recruitment of TRAFs. Among the TRAF family of proteins, TRAF6 is essential for RANKL-induced osteoclastogenesis. TRAF6 activates downstream signaling components such as NF-κB and MAPK. RANKL promotes osteoclast differentiation via activation of the NF-κB pathway. RANK signaling also activates the transcription factor AP-1 by inducing its component c-Fos. Activation of NF-κB and c-Fos leads to the induction of NFATc1, which stimulates the expression of osteoclast-specific genes such as cathepsin K. RANKL also induces the phosphorylation of MITF, which is another transcription factor involved in osteoclastogenesis that is activated downstream of MAPK. The transcription factor complex, including transcription factors such as NFATc1 and MITF, stimulates the expression of osteoclast-specific genes such as cathepsin K. In osteoclastogenesis, the expression of NFATc1 is strongly induced via autoamplification in the later stages of differentiation. Transcriptional activity of NFATc1 is dependent on the localization of protein in nuclei, which is regulated by calcium signaling. Costimulatory molecules possessing tyrosine-based activation motif-harboring adaptors induce calcium signaling in osteoclastogenesis. In addition, RANK has a unique cytoplasmic domain that recruits adaptor proteins Gab2 and PLCγ2, which also induce calcium signaling. Phosphorylation of PLCγ2 in conjunction with costimulatory signals lead to the activation of NFATc1. NFATc1 also stimulates transcription of the D2 isoform of the vacuolar ATPase Vo domain and of the dendritic cell-specific transmembrane protein DC-STAMP, which are involved in multinucleation of osteoclasts. Thus, it is generally accepted that NFATc1 is involved in the differentiation and multinucleation of osteoclasts.
During bone resorption, osteoclasts attach to the bone surface, acidify and solubilize the mineral portion of the matrix, and degrade the organic matrix of bone by proteinases such as cathepsin K in the sealed bone–cell interface. Recently, another transcription factor, LRF (also previously known as OCZF in rats and FBI-1 in humans), was shown to be involved in bone resorption activity of osteoclasts. LRF (also called POKEMON), previously reported to be proto-oncogene, is a regulator of differentiation of hematopoietic cells including B and T cells, and erythrocytes. OCZF is expressed in mature osteoclasts in vivo, and the expression is induced by RANKL in osteoclastogenesis in vitro. LRF together with NFATc1 increases the transcriptional activity of cathepsin K, thereby increasing bone resorption. Activation of bone resorption involves polarization of osteoclasts. Polarization requires activation of c-Src by integrin αvβ3, which induces reorganization of the cytoskeleton. It was recently shown that RANKL directly affects this process. Treatment of prefusion osteoclasts with RANKL induces c-Src to associate with RANK and cytoskeletal-organizing molecules such as Syk, Vav3 and Rac1. In osteoclasts, Rho GTPases such as RhoA, Rac1 and Cdc42 control the organization of F-actin, and the vesicular trafficking pathway is regulated by Rab7, which is a GTPase member of the Rab family.
Integrin-mediated signaling in osteoclasts, which is also important for the survival of osteoclasts, is regulated by several cytokines including RANKL. Downstream of RANK, c-Src activates PI3K and AKT, which subsequently phosphorylates BAD and inactivates caspase 9, thereby preventing activation of the apoptosis cascade. JNK and NF-κB activated by RANKL increase the expression of antiapoptosis genes, such as Bcl-2 and Bcl-xl. RANKL also reduces the level of Fas expression in mature osteoclasts, thereby reducing Fas-mediated apoptosis. Osteoclasts express TRAIL, which is involved in osteoclast apoptosis. OPG is a decoy receptor for RANKL, but also binds and inhibits TRAIL. By inhibiting TRAIL, OPG directly decreases apoptosis of osteoclasts.
The action of RANKL/RANK/OPG in osteoclastogenesis is influenced by the microenvironment. Extracellular matrix components, such as glycosaminoglycans (GAGs) including hyaluronan and heparin, affect osteoclastogenesis. Heparin and GAGs inhibit adherence and spreading of osteoclast precursors and inhibit osteoclast differentiation. By contrast, heparin enhances bone resorption of osteoclasts by inhibiting OPG function in the C-terminal heparin binding domain. It has been reported that GAGs highly expressed in osteosarcoma may regulate RANKL function by inhibiting RANKL–OPG interactions.
Source...