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Osteoprotegerin ligand: A common link between osteoclastogenesis, lymph node formation and lymphocyte development
Abstract
The TNF-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL or ODF) has been identified as the osteoclast differentiation factor and a regulator of T cell-dendritic cell interactions in the immune system. Surprisingly, the same molecule was identified as a crucial factor in early lymphocyte development and lymph node organogenesis. We will discuss the role of OPGL in bone remodelling and the immune system.
... In addition, RANKL also plays a regulatory role in interaction of T-cells and dendritic cells in the immune system. This contributes significantly to initial lymphocyte development and lymph node formation [20]. In fact, studies indicate that RANKL deficient mice devel- ...
... Vitamin D, PTH, glucocorticoids as well as PGE 2 increase RANKL expression and downregulates OPG production, which leads to an increase in RANK-RANKL interaction, thereby leading to osteoclastogenesis and increase in activity of osteoclasts [8,20]. PGE 2 is increased in inflammatory processes due cytokine IL-1 production and in turn promotes osteoclastic bone resorption through above-mentioned action [28]. ...
Osteoporosis is a common clinical pathology encountered in everyday medicine and while the foremost population that comes to mind is elderly postmenopausal women, it may affect varied age-groups and indeed, has several causative factors. One such interesting association is between gastrointestinal illnesses like IBD and osteoporosis. Osteoporosis is important to diagnose because it increases the likelihood of fractures and in IBD patients, this should be especially noted because they are often on glucocorticoid therapy, which on its own is responsible for an increase in fragility fractures. With age and decrease in estrogen, the loss of bone mineral density increases. As a result, patients are at an increased risk of developing osteoporosis and subsequent fractures. In IBD subjects, paramount aspect is the early onset of osteopenia and osteoporosis. Hence, guiding us towards the understanding that in addition to age, genetics, and lifestyle, there are a number of factors operating at a molecular and nutritional status level that lead to osteoporosis. This review article discusses the epidemiology and pathophysiology of osteoporosis in IBD patients to help garner an in-depth view of the imbalance in the several factors that play a role in the pathogenesis and provide a composite view of this common association.
... 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. ...
... 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. ...
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.
... Binding of RANK to RANKL induces a signalling and gene expression cascade that results in the differentiation and maturation of osteoclasts. OPG blocks the interaction, thereby inhibiting osteoclast formation [27]. Therefore, factors that increase RANKL enhance osteoclastogenesis, whereas factors that increase OPG inhibit osteoclastogenesis. ...
... This system may be critical in linking systemic or mucosal inflammation with altered bone metabolism and, ultimately, osteoporosis. We found nonsignificantly higher levels of OPG and sRANKL in IBD patients at baseline compared with controls, and these results are consistent with the findings of previous studies [19,23,27,28]. These studies found a higher concentration of OPG in active CD patients compared with controls [26,29] and also decreased OPG levels following IFX therapy [19]. ...
Objectives:
Infliximab (IFX) treatment has shown potentially beneficial effects on bone metabolism in inflammatory bowel disease (IBD) patients. We aimed to prospectively evaluate the impact of IFX treatment on bone metabolism in antitumour necrosis factor (TNF)-α-naive IBD patients using established bone metabolism markers and an in-vitro osteoblast model.
Materials and methods:
A total of 37 anti-TNFα-naive IBD patients and 20 healthy controls were included. All measurements were performed at baseline and repeated in IBD patients following IFX therapy. Bone mineral density was measured by dual-energy X-ray absorptiometry. Parathyroid hormone, vitamin D, osteoprotegerin, soluble receptor activator of nuclear factor B ligand and proinflammatory and anti-inflammatory cytokines were measured. Bone formation was measured using osteocalcin (OC) and procollagen type 1N propeptide, and bone resorption was measured using serum type 1 collage c-telopeptide. The effect of control and IBD patient sera on human osteoblast viability and differentiation was analysed.
Results:
OC level was higher in controls than IBD patients (P=0.018). After IFX, OC and procollagen type 1N propeptide increased significantly (P=0.002 and 0.011) and (P<0.001 and P=0.016) at weeks 6 and 30 after treatment, respectively. There was a nonsignificant decrease in serum type 1 collage c-telopeptide. After IFX therapy, proinflammatory cytokines TNF-α, interleukin-6 and interleukin-13 decreased significantly (P=0.016, week 54; P=0.005, week 6 and P=0.025, week 6), respectively. Sera from IBD patients before IFX showed increased osteoblast viability compared with the controls (P=0.003 to P<0.005), but induced reduced osteoblast differentiation. After IFX, viability reduced to control levels, but osteoblast differentiation increased (P=0.041).
Conclusion:
IFX treatment induced beneficial effects on bone metabolism. Osteoblast culture results suggest that IBD patients may have increased osteoblast viability, but reduced differentiation, which has implications for bone strength.
... migration, and differentiation of the myeloid progenitors. The differentiation of OCs is induced by two crucial determinants: M-CSF, which is essential for OC survival, and RANKL, which acts as a differentiation factor of OCs [4,5]. The binding of RANKL to its receptor RANK recruits tumor necrosis factor (TNF)-receptor-associated factors (TRAFs), which then activate downstream signaling molecules including mitogen-activated protein kinases (MAPKs), NF-κB, and activator protein-1 (AP-1), leading to the activation of the nuclear factor of activated T cells 1 (NFATc1) [3]. ...
Bone homeostasis is regulated by the balanced actions of osteoblasts that form the bone and osteoclasts (OCs) that resorb the bone. Bone-resorbing OCs are differentiated from hematopoietic monocyte/macrophage lineage cells, whereas osteoblasts are derived from mesenchymal progenitors. OC differentiation is induced by two key cytokines, macrophage colony-stimulating factor (M-CSF), a factor essential for the proliferation and survival of the OCs, and receptor activator of nuclear factor kappa-B ligand (RANKL), a factor for responsible for the differentiation of the OCs. Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases, play an essential role in regulating the proliferation, differentiation, and function of OCs. ERKs have been known to play a critical role in the differentiation and activation of OCs. In most cases, ERKs positively regulate OC differentiation and function. However, several reports present conflicting conclusions. Interestingly, the inhibition of OC differentiation by ERK1/2 is observed only in OCs differentiated from RAW 264.7 cells. Therefore, in this review, we summarize the current understanding of the conflicting actions of ERK1/2 in OC differentiation.
... Indeed, such a mechanism might avoid possible off-target effects by localizing the increased RANK-EV levels to the bone microenvironment being remodeled. Because the RANKL/RANK signaling network also plays an important role in communication between immune cells [17,18] , off-target effects are a real concern if RANK-EV levels are increased systemically. Unfortunately, nothing is known about the subset of EVs from osteoclasts that contain RANK, so there is no basis for rational strategies to stimulate RANK-EV production. ...
Aim: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail.
Methods: The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs.
Results: Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast’s cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81.
Conclusion: RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.
... 143 The NF-κB signaling pathway is essential for OC formation and bone resorption (Fig. 5). The NF-κB pathway is mediated through an upstream RANKL-RANK combination, which activates downstream signaling such as NF-κB, c-Fos, and NFATc1 signaling, [144][145][146][147][148][149] contributing to inhibited osteogenic differentiation of BMSCs and differentiated pre-OCs into OCs. Moreover, Yamashita et al. confirmed that NF-κB p50 and p52 regulated receptor activator of NF-κB ligand (RANKL) by activating c-Fos and NFATc1. ...
Numerous small-molecule amines (SMAs) play critical roles in maintaining bone homeostasis and promoting bone regeneration regardless of whether they are applied as drugs or biomaterials. On the one hand, SMAs promote bone formation or inhibit bone resorption through the regulation of key molecular signaling pathways in osteoblasts/osteoclasts; on the other hand, owing to their alkaline properties as well as their antioxidant and anti-inflammatory features, most SMAs create a favorable microenvironment for bone homeostasis. However, due to a lack of information on their structure/bioactivity and underlying mechanisms of action, certain SMAs cannot be developed into drugs or biomaterials for bone disease treatment. In this review, we thoroughly summarize the current understanding of SMA effects on bone homeostasis, including descriptions of their classifications, biochemical features, recent research advances in bone biology and related regulatory mechanisms in bone regeneration. In addition, we discuss the challenges and prospects of SMA translational research.
... IL1b has been demonstrated to inhibit OPG levels and increase RANKL levels in osteoblasts (89). IL1b and IL8 modulate the OPG-RANKL-RANK system and increase bone resorption (90). Finally, IL1b upregulates IL8 and OPG in primary human gingival fibroblasts and periodontal ligament fibroblasts (91). ...
Several periodontitis-associated species induce genes related to cell proliferation, cell-cycle progression, apoptosis, intracellular transport, immune and inflammatory responses, carcinogenesis, and cancer progression. Here, we reviewed studies showing strong associations between gingival squamous cell carcinoma (GSCC) and three periodontitis-related oral bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia) in the microenvironment. These bacteria produce cytoplasmic lipopolysaccharide (LPS) and secrete cytokines and molecules related to carcinogenesis and tumor progression and metastasis. In GSCC, LPS levels are substantially elevated and assessable by functional analysis. Polymicrobial infections can synergistically induce inflammation and significantly upregulate proinflammatory molecules (e.g., interleukin [IL]-1β, IL-8, and tumor necrosis factor-α) that promote carcinogenesis. This review can facilitate the identification of novel targets for treating and preventing GSCC which is potentially associated with periodontal pathogen.
... A central and essential regulatory network in bone remodeling involves receptor activator of nuclear factor kappa B (RANK) and RANK-Ligand (RANKL) (Boyce & Xing, 2008;Honma et al., 2020;Lacey et al., 1998). RANKL, a transmembrane protein found on osteocytes and osteoblasts, stimulates RANK found on osteoclasts and their precursors, initiating regulatory pathways resulting in osteoclastogenesis, osteoclast survival and osteoclast bone resorptive activity (Kong et al., 1999;Lacey et al., 1998;Martin & Sims, 2015;Yasude et al., 1998). Recent data showed that RANK is found in extracellular vesicles (EVs) shed by osteoclasts (Huynh, 2016). ...
Osteoporosis results in over a million bone fractures each year despite current therapeutics that inhibit bone resorption. The need for better anti-osteoporotic therapeutics is clear. Recently, extracellular vesicles (EVs) containing RANK that are released by osteoclasts and serve as novel regulators of bone remodeling were identified. These RANK-containing EVs both block bone resorption by osteoclasts and stimulate a RANKL reverse signaling pathway in osteoblasts that promotes bone formation. Small molecules with the same activity could prove to be a new class of therapeutic for treatment of bone disease. To seek such small molecules, a virtual screen identified small molecules that were predicted to bind RANKL in the same location as RANK, and an initial cell culture screen showed eighteen of the candidates reduced osteoclast formation in vitro at a concentration of 100 μM. In this project, one candidate, 3-Nitro-4-phosphobenzoic acid (NPA), was examined in greater detail. NPA does-dependently reduced recombinant RANKL-stimulated differentiation of RAW 264.7 cells into osteoclast-like cells with an IC50 of 38 μM. In calcitriol-stimulated mouse marrow, primary cultures that produce both osteoblasts and osteoclasts, NPA (50 μM) reduced osteoclast formation by 88%, while alkaline phosphatase positive osteoblast numbers were higher. These data show that NPA, in cell culture, is able to simultaneously inhibit osteoclast differentiation and promote osteoblast formation. Thus, NPA is a candidate to be a lead molecule for novel dual-function, small molecule, therapeutic agents to treat osteoporosis.
... In vivo evidence for metformin anti-inflammatory mode of action was obtained in a mouse model of rheumatoid arthritis (Fan et al., 2020) where it primarily affected IL-1β. Together with the downregulated crucial osteoclastogenesis factor RANKL (Kong et al., 1999) (Figure 3G), it seems likely that also other inflammatory osteoclastogenesis genes could be affected. Downregulation of RANKL was also seen in ovariectomized rats that were treated with metformin (Mai et al., 2011), who also showed that this brought down the number of osteoclasts in vivo. ...
Diabetes and periodontitis are comorbidities and may share common pathways. Several reports indicate that diabetes medication metformin may be beneficial for the periodontal status of periodontitis patients. Further research using appropriate cell systems of the periodontium, the tissue that surrounds teeth may reveal the possible mechanism. Periodontal ligament fibroblasts anchor teeth in bone and play a role in the onset of both alveolar bone formation and degradation, the latter by inducing osteoclast formation from adherent precursor cells. Therefore, a cell model including this type of cells is ideal to study the influence of metformin on both processes. We hypothesize that metformin will enhance bone formation, as described for osteoblasts, whereas the effects of metformin on osteoclast formation is yet undetermined. Periodontal ligament fibroblasts were cultured in the presence of osteogenic medium and 0.2 or 1 mM metformin. The influence of metformin on osteoclast formation was first studied in PDLF cultures supplemented with peripheral blood leukocytes, containing osteoclast precursors. Finally, the effect of metformin on osteoclast precursors was studied in cultures of CD14 ⁺ monocytes that were stimulated with M-CSF and receptor activator of Nf-κB ligand (RANKL). No effects of metformin were observed on osteogenesis: not on alkaline phosphatase activity, Alizarin red deposition, nor on the expression of osteogenic markers RUNX-2, Collagen I and Osteonectin. Metformin inhibited osteoclast formation and accordingly downregulated the genes involved in osteoclastogenesis: RANKL, macrophage colony stimulating factor (M-CSF) and osteoclast fusion gene DC-STAMP. Osteoclast formation on both plastic and bone as well as bone resorption was inhibited by metformin in M-CSF and RANKL stimulated monocyte cultures, probably by reduction of RANK expression. The present study unraveling the positive effect of metformin in periodontitis patients at the cellular level, indicates that metformin inhibits osteoclast formation and activity, both when orchestrated by periodontal ligament fibroblasts and in cytokine driven osteoclast formation assays. The results indicate that metformin could have a systemic beneficiary effect on bone by inhibiting osteoclast formation and activity.
... The discovery of RANKL in EVs in 2015 [14] , and RANK in EVs in 2016 [15] , together with other recent advances in the understanding of bone biology, particularly the finding that most RANKL that stimulates bone resorption is from osteocytes [16][17][18][19][20] , has opened up new lines of study [ Figure 1]. Incorporation of these transmembrane proteins into EVs allows each to be, as needed, either a receptor or a ligand, serving as an Figure 1. ...
Receptor activator of nuclear factor kappa B-ligand (RANKL), its receptor RANK, and osteoprotegerin which binds RANKL and acts as a soluble decoy receptor, are essential controllers of bone remodeling. They also play important roles in establishing immune tolerance and in the development of the lymphatic system and mammary glands. In bone, RANKL stimulates osteoclast formation by binding RANK on osteoclast precursors and osteoclasts. This is required for bone resorption. Recently, RANKL and RANK have been shown to be functional components of extracellular vesicles (EVs). Data linking RANKL and RANK in EVs to biological regulatory roles are reviewed, and crucial unanswered questions are examined. RANKL and RANK are transmembrane proteins and their presence in EVs allows them to act at a distance from their cell of origin. Because RANKL-bearing osteocytes and osteoblasts are often spatially distant from RANK-containing osteoclasts in vivo, this may be crucial for the stimulation of osteoclast formation and bone resorption. RANK in EVs from osteoclasts has the capacity to stimulate a RANKL reverse signaling pathway in osteoblasts that promotes bone formation. This serves to couple bone resorption with bone formation and has inspired novel bifunctional therapeutic agents. RANKL- and RANK- containing EVs in serum may serve as biomarkers for bone and immune pathologies. In summary, EVs containing RANKL and RANK have been identified as intercellular regulators in bone biology. They add complexity to the central signaling network responsible for maintaining bone. RANKL- and RANK-containing EVs are attractive as drug targets and as biomarkers.
... LPSinduced pro-in ammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, prostaglandin E2 (PGE2) and IL-6, can directly stimulate osteoclast differentiation and ultimately lead to the destructive bone loss by means of increasing the expression of receptor activator for nuclear factor-κB ligand (RANKL) [12][13][14]. Furthermore, osteoclasts arise in the bone marrow from the fusion of haematopoietic cells of a monocyte/macrophage lineage after stimulation by M-CSF and RANKL [15]. ...
Background
Lipopolysaccharide (LPS), a bacteria product, plays an important role in orthopedic diseases. Drugs that inhibit LPS-induced osteoclastogenesis are urgently needed for the prevention of bone destruction.Methods
In this study, we evaluated the effect of β-ecdysterone (β-Ecd), a major component of Chinese herbal medicines derived from the root of Achyranthes bidentata BI on LPS-induced osteoclastogenesis in vitro and explored the mechanism underlying the effects of β-Ecd on this.ResultsWe showed that β-Ecd inhibited LPS-induced osteoclast formation from osteoclast precursor RAW264.7 cells. The inhibition occurred through suppressing the production of osteoclast activating TNF-α, IL-1β, PGE2 and COX-2, which led to down-regulating expression of osteoclast-related genes including RANK, TRAF6, MMP-9, CK and CAⅡ. Besides, β-Ecd treatment can inhibit LPS-induced activation of NF-κB signaling pathway in RAW264.7 cells. Meanwhile, inhibition of NF-κB signaling pathway decreased the formation of osteoclasts and expression of pro-inflammatory cytokines which LPS-induced. Collectively, β-Ecd can prevent LPS-induced osteoclast formation in vitro by regulating NF-κB signaling pathway.ConclusionsThese findings provide evidences that β-Ecd might be beneficial as a valuable choice for the prevention and treatment of bacteria-induced bone destruction disease, and give new insights for understanding its possible mechanism.
... During bone metastasis of PCa, cancer cell-derived cytokines stimulate expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which in turn activates bone resorption. RANKL, also known as tumour necrosis factor-related activation-induced cytokine (TRANCE) [11], osteoprotegerin ligand (OPGL) [12,13] and osteoclastic differentiation factor (ODF) [14], interacts with RANK and is involved in all the steps related to tumour development, from initial tumour formation to migration of cancer cells and subsequent metastasis [15]. RANKL is expressed in several tissues, including brain, skin, intestine, skeletal muscle, kidney, liver, lung and mammary tissue; however, expression is very high in bone [16], lymphoid organs and the vascular system [17]. ...
Background: Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. RANKL was associated with epithelial-mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments.
Method: In this study, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa.
Results: RANKL activation was observed in human PCa tissue specimens. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis.
Conclusion: Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa
Trial registration: Chosun University Hospital, CHOSUN 2020-06-001. Registered 01 June 2020-prospectevely registered, https://hosp.chosun.ac.kr/medi_depart/ site=hospital&mn=151&type=view&catename=IRB
... During bone metastasis of PCa, cancer cell-derived cytokines stimulate expression of receptor activator of nuclear factor kappa-B ligand (RANKL), which in turn activates bone resorption. RANKL, also known as tumour necrosis factor-related activation-induced cytokine (TRANCE) [11], osteoprotegerin ligand (OPGL) [12,13] and osteoclastic differentiation factor (ODF) [14], interacts with RANK and is involved in all the steps related to tumour development, from initial tumour formation to migration of cancer cells and subsequent metastasis [15]. RANKL is expressed in several tissues, including brain, skin, intestine, skeletal muscle, kidney, liver, lung and mammary tissue; however, expression is very high in bone [16], lymphoid organs and the vascular system [17]. ...
Background: Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. RANKL was associated with epithelial-mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments.
Method: In this study, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa.
Results: RANKL activation was observed in human PCa tissue specimens. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis.
Conclusion: Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa
Trial registration: Chosun University Hospital, CHOSUN 2020-06-001. Registered 01 June 2020-prospectevely registered, https://hosp.chosun.ac.kr/medi_depart/?site=hospital&mn=151&type=view&catename=IRB
... 90,91 In fact, previous studies considering that the binding of the M-CSF and c-Fms induced signalling pathways required for osteoclastic precursor survival and proliferation, 92 whereas the binding of RANKL and RANK conducted signalling cascades required for differentiation of osteoclastic precursors and the resorptive function of matured osteoclast. 93 In that, M-CSF interact with its cognate receptor c-Fms could lead the specific tyrosine residues autophosphorylation and transphosphorylation in the site of cytoplasmic tail of c-Fms. 94 However, among the c-Fms cytoplasmic tail tyrosine residues, four crucial tyrosine residues (including: Y559, Y697, Y721 and Y921) participate the regulation of osteoclastic precursors survival and proliferation. ...
In the ageing skeleton, the balance of bone reconstruction could commonly be broken by the increasing of bone resorption and decreasing of bone formation. Consequently, the bone resorption gradually occupies a dominant status. During this imbalance process, osteoclast is unique cell linage act the bone resorptive biological activity, which is a highly differentiated ultimate cell derived from monocyte/macrophage. The erosive function of osteoclasts is that they have to adhere the bone matrix and migrate along it, in which adhesive cytoskeleton recombination of osteoclast is essential. In that, the podosome is a membrane binding microdomain organelle, based on dynamic actin, which forms a cytoskeleton superstructure connected with the plasma membrane. Otherwise, as the main adhesive protein, integrin regulates the formation of podosome and cytoskeleton, which collaborates with the various molecules including: c‐Cbl, p130Cas, c‐Src and Pyk2, through several signalling cascades cross talking, including: M‐CSF and RANKL. In our current study, we discuss the role of integrin and associated molecules in osteoclastogenesis cytoskeletal, especially podosomes, regulation and relevant signalling cascades cross talking.
... OPG and RANKL are considered as members of a ligandreceptor system that controls osteoclast differentiation and bone resorption . RANKL not only activates mature osteoclasts but also mediates the process of osteoclastogenesis in the presence of M-CSF (Kong et al. 1999). Soluble and membranous form of RANKL is expressed by preosteoblastic cells but their specific receptor RANK is located on the osteoclast progenitor cells (Lacey et al. 1998). ...
... cathelicidin and serum amyloid A, generic inflammatory markers with anti-microbial roles in Atlantic salmon (Chang et al., 2006;Jensen et al., 1997;Lee et al., 2017). Irg1 or cis-aconitate decarboxylase contributes to the antimicrobial activity of macrophages (Michelucci et al., 2013), and mammalian tnfrsf11b is a regulator of lymphocyte development (Kong et al., 1999). The increased expression of innate response genes in vaccinated fish persisted for at least four weeks and many genes maintained an activated status to the end of observations. ...
The majority of studies of vaccine responses in Atlantic salmon have focused on several weeks after vaccination, and employed a limited number of marker genes. In this study, novel techniques were used to examine a broad panel of expressed genes and antibody repertoire of Atlantic salmon following vaccination. Salmon parr were vaccinated with a multivalent oil-based vaccine, and blood plasma and head kidney were sampled at several time-points between 0–35 days post vaccination. Saline-injected fish were used as control at all time-points. Microarray analyses showed increased expression of immune genes from the first day to the end of study in the head kidney of vaccinated fish. Genes up-regulated in the late phase included several leukocyte markers and components of the oxidative burst complex. A suite of genes that can take part in B cells differentiation were up-regulated from day 14, at which time secretory IgM transcripts also peaked. This coincided with marked increased plasma titres of non-vaccine specific antibodies binding to a hapten-carrier antigen DNP-KLH, while antibodies to bacterial components of the vaccine, Moritella viscosa and Aeromonas salmonicida, first showed significantly elevated antibody levels at day 21, and at a markedly lower magnitude than the non-vaccine specific titres. Sequencing of the variable region of IgM heavy chain (CDR3) revealed higher cumulative frequencies of unique clonotypes in vaccinated salmon starting from day 14 when specific antibodies were first detected. Reduced sequence variance of CDR3 suggested expansion of recently emerged clonotypes. Overall, the results presented here follow a broad panel of gene expression, immunoglobulin sequencing and plasma antibody titres in the first few weeks after vaccination of Atlantic salmon, pointing to a potentially important contribution of non-vaccine specific antibody responses early in the vaccine response.
... Since ALP is an early indicator of osteoblast differentiation and COLA1 is expressed prior to ALP, this trend is expected. OPG, as a decoy RANKL receptor, also enhances differentiation and absence of OPG has been linked to decreased bone mass in mice 34,35 . In this study, OPG expression was higher with 0.25 Cu-TCP compared to Pure-TCP at later kinetic. ...
Tricalcium phosphate (TCP) is a promising candidate in bone and dental tissue engineering applications. Though osteoconductive, its low osteoinductivity is a major concern. Trace elements addition at low concentrations are known for their impact on not only the osteoinductivity, but also physical and mechanical properties of TCP. Copper (Cu) is known for its role in vascularization and angiogenesis in biological systems. Here, we studied the effects of Cu addition on phase composition, porosity, microstructure and in vitro interaction with osteoblast (OB) cells. Our results showed that Cu stabilized the TCP structure, while no significant effect of microstructure and porosity was found. Cu at concentrations less than 1 wt.% did not have any cytotoxic effect while decreased proliferation of OBs were observed at 1 wt.% Cu doped TCP. Addition of Cu upregulated collagen type I and vascular endothelial growth factor expression in a dose dependent manner at early time-point. Furthermore, Cu reduced inflammatory gene expression by human osteoblasts. These findings show that addition of Cu to TCP may provide a therapeutic strategy that can be applied in bone tissue engineering applications.
... The interaction of RANKL or RANK is modified by other factors produced by osteoblasts, called as osteoprotegerin (OPG), a soluble feed receptor for RANKL [23]. Osteoprotegerin prevents RANKL from binding to RANK, so OPG suppresses bone resorption and maintains a balance between bone resorption and bone formation [24]. ...
Mechanical force of orthodontics, would inhibit periodontal ligament vascularization and blood flow, causing biochemical and cellular changes, such as cell deformation, inflammation, and circulatory disturbances. Each of these conditions afecting cell diferentiation, cell repair, and cell migration, is driven by numerous molecular and inflammatory mediators. Fibroblasts, osteoblasts, osteocytes, osteoclasts, odontoblasts, cementoblasts, chondrocytes and immune cells are the major cell types involved in the remodeling process on orthodontic tooth movement. Hyperbaric oxygen therapy is one of many solutions which stimulates the growth of new blood vessels and result in a substantial increase in tissue oxygenation. It plays a role in bone remodeling process.Purpose: To determine the differences of Hyperbaric Oxygen (HBO)2.4 ATA 7 and 10 days in osteoblast and osteocytes number during bone remodelling in Orthodontic tooth movement. Materials and Methods: The experiment using a post test only control group design. 32 male guinea pigs were randomly divided into 4 groups. K1 was control group without any treatment, K2 was a group which was given a mechanical orthodontic pressure, K3 was the group treated mechanical orthodontic with the addition of hyperbaric oxygen therapy. The maxillary incisors were moved distally by elastic separator. After HBO on day 7, all groups were sacrificed then analyzed osteoblast and osteocytes number by One-way ANOVA and LSD statistical test. Results: The study showed significant differences in osteoblast and osteocytes number during bone remodelling in orthodontic tooth movement between groups.Conclusion: HBO therapy 2.4 ATA for 7 days effective to induce bone remodelling during orthodontic tooth movement.
... This may account for much higher levels of alveolar bone remodeling than root surface remodeling during orthodontic tooth movement. Studies have demonstrated that during orthodontic tooth movement, the OPG/RANKL system plays an important role in the resorption and remodeling of alveolar bone [22][23][24]. ...
Purpose: To investigate the mechanism involved in the anti-resorptive effect of cementocytes during orthodontic tooth movement in mice and human specimens. Methods: The morphology, molecular structure and biological expression of cellular cementum in mice and human samples were examined using hematoxylin and eosin staining, immuno-histochemical staining, scanning electron microscopy, and Raman spectroscopy. The expressions of osteoprotegerin (OPG), receptor activator of nuclear κB ligand (RANKL) and sclerostin (SOST) encoding genes in cementocytes and alveolar bone osteocytes were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Results: Results demonstrated that cementocyte lacunae were larger and more irregular in shape than the regular ellipsoid osteocyte lacunae. The ratio of phosphate to amino acid was significantly lower in cellular cementum than that in alveolar bone and dentin. In mice, OPG/RANKL ratio was significantly higher in cementocytes (4.8 ± 0.37) than in alveolar bone osteocytes (0.17 ± 0.42) in natural state. In humans, OPG/RANKL ratio was 1.41 ± 0.07 in cementocytes and 0.71 ± 0.04 in alveolar bone osteocytes under natural conditions, and 37.69 ± 0.15 in cementocytes and 1.95 ± 0.83 in alveolar bone osteocytes applying fluid flow shear stress. Moreover, SOST was extremely low expressed under force application in cementocytes. Conclusion: Under fluid flow sheer stress, cementocytes stimulate the differentiation of osteoblasts and inhibit the activation of osteoclasts, showing greater potential for bone protection than alveolar bone osteocytes. Cementocytes might play an important role in preventing root resorption in the process of orthodontic tooth movement. © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria and 2018 The authors.
... RANKL (TNFSF11) signalization is implicated in the development, histogenesis, and functional homeostasis of various tissues, particularly lymphoid tissues, skin appendages (hair, teeth, and mammary glands) and skeletal components [1][2][3][4][5][6][7][8]. During development, expression of RANKL, as well as expressions of its receptors RANK and OPG, have been reported in the spleen [9], thymus [10,11], lymph nodes [12,13], hair [14], teeth [15,16], mammary glands [17,18], and bones, regardless of the ossification process involved: endochondral [19][20][21] or intramembranous [19,20]. ...
RANKL signalization is implicated in the morphogenesis of various organs, including the skeleton. Mice invalidated for Rankl present an osteopetrotic phenotype that was less severe than anticipated, depending on RANKL’s implication in morphogenesis. The hypothesis of an attenuated phenotype, as a result of compensation during gestation by RANKL of maternal origin, was thus brought into question. In order to answer this question, Rankl null mutant pups from null mutant parents were generated, and the phenotype analyzed. The results validated the presence of a more severe osteopetrotic phenotype in the second-generation null mutant with perinatal lethality. The experiments also confirmed that RANKL signalization plays a part in the morphogenesis of skeletal elements through its involvement in cell-to-cell communication, such as in control of osteoclast differentiation. To conclude, we have demonstrated that the phenotype associated with Rankl invalidation is attenuated through compensation by RANKL of maternal origin.
... 56 What is of importance here is that the biological activity of OPG is not limited solely to bone tissue but is also expressed and acts in other cells and tissue types, eg, T lymphocytes and large arteries where OPG may play an important role in both development and function of these cells and tissues. 62 Regarding metabolic syndromes, an attempt at elucidation of the possible function of OPG in the cross talk between NAFLD and bone tissue was made by Yilmaz et al. They have shown that in patients with NASH and borderline NASH, serum OPG concentrations are reduced in patients with more severe forms of NAFLD and may serve as a noninvasive biomarker to identify patients with NASH. ...
Rafał Filip,1,2 Radosław P Radzki,3 Marek Bieńko3 1Department of Gastroenterology with IBD Unit, Clinical Hospital 2, Rzeszów, Poland; 2University of Rzeszów, Rzeszów, Poland; 3Department of Animal Physiology, University of Life Sciences, Lublin, Poland Abstract: Excess fat deposition and insulin resistance are considered the main risk factors for nonalcoholic fatty liver disease (NAFLD), and therefore, not surprisingly, the global prevalence of NAFLD increases in parallel with both obesity and type 2 diabetes. Although deterioration of bone homeostasis in patients with NAFLD is commonly observed, its etiology has not been fully elucidated yet. It was shown in several studies that bone tissue seems to be independently associated with NAFLD. A mechanistic perspective puts the liver at the center of mutual interdependencies obviously involving adipose tissue and muscles and also the bone matrix and bone cells, which are relatively novel. In this review, various pathophysiological mechanisms and possible mediating molecules that may interplay between NAFLD and bone tissue are described. Chronic inflammation, vitamin D3, growth hormone, insulin-like growth factor 1, osteopontin, fetuin-A, irisin, osteocalcin, and osteoprotegerin from osteoblasts have been proposed as mediators of mutual interactions among the skeleton, fatty tissue, and liver. Although to date there are still many issues that have not been elucidated, growing evidence suggests that screening and surveillance of bone mineral density in patients with NAFLD should be considered in future strategies and guidelines for NAFLD management. Keywords: nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, inflammation, osteoporosis, bone metabolism
... Despite the expected modulation of bone resorption, untreated and anti-RANKL-treated lesions presented similar immuneinflammatory features at early active stages (Garlet et al. 2012;Araujo-Pires et al. 2014). In addition, despite reducing bone destruction, RANKL inhibition did not modify the local inflammation microenvironment in experimental arthritis (Kong et al. 1999;Stolina et al. 2009;Ferrari-Lacraz and Ferrari 2011). However, our results demonstrate that RANKL inhibition was associated with persistent inflammation over time, with gene expression profiles compatible with an active lesion phenotype (Araujo-Pires et al. 2014;Francisconi et al. 2016). ...
The chronic inflammatory immune response triggered by the infection of the tooth root canal system results in the local upregulation of RANKL, resulting in periapical bone loss. While RANKL has a well-characterized role in the control of bone homeostasis/pathology, it can play important roles in the regulation of the immune system, although its possible immunoregulatory role in infectious inflammatory osteolytic conditions remains largely unknown. Here, we used a mouse model of infectious inflammatory periapical lesions subjected to continuous or transitory anti-RANKL inhibition, followed by the analysis of lesion outcome and multiple host response parameters. Anti-RANKL administration resulted in arrest of bone loss but interfered in the natural immunoregulation of the lesions observed in the untreated group. RANKL inhibition resulted in an unremitting proinflammatory response, persistent high proinflammatory and effector CD4 response, decreased regulatory T-cell (Treg) migration, and lower levels of Treg-related cytokines IL-10 and TGFb. Anti-RANKL blockade impaired the immunoregulatory process only in early disease stages, while the late administration of anti-RANKL did not interfere with the stablished immunoregulation. The impaired immunoregulation due to RANKL inhibition is characterized by increased delayed-type hypersensitivity in vivo and T-cell proliferation in vitro to the infecting bacteria, which mimic the effects of Treg inhibition, reinforcing a possible influence of RANKL on Treg-mediated suppressive response. The adoptive transfer of CD4+FOXp3+ Tregs to mice receiving anti-RANKL therapy restored the immunoregulatory capacity, attenuating the inflammatory response in the lesions, reestablishing normal T-cell response in vivo and in vitro, and preventing lesion relapse upon anti-RANKL therapy cessation. Therefore, while RANKL inhibition efficiently limited the periapical bone loss, it promoted an unremitting host inflammatory response by interfering with Treg activity, suggesting that this classic osteoclastogenic mediator plays a role in immunoregulation.
... Besides its role in bone homeostasis, RANKL has been shown to be implicated in the mammary gland development, in the immune system regulation and as a survival factor for dendritic cells [10][11][12]. Looking at the pathologic side, its role as a pro-tumour and pro-metastatic factor in breast cancer has also been clearly ascertained. As it will be described later, tumour cells produce RANKL, which acts in an autocrine manner and in support of the osteomimicry profile. ...
Metastasis is defined as a very inefficient process, since less than 0.01% of cancer cells injected into the circulation will engraft in a distant organ, where they must acquire the ability to survive and proliferate inside a “foreign” environment. In bone metastases, the interaction with the host organ is much more favoured if tumour cells gain “osteomimicry”, that is the ability to resemble a resident bone cell (i.e. the osteoblast), thus intruding in the physiology of the bone. This is accomplished by the expression of osteoblast markers (e.g. alkaline phosphatase) and the production of bone matrix proteins and paracrine factors which deregulate the physiology of bone, fuelling the so-called “vicious cycle”. The main challenge of researchers is therefore to identify the genetic profile determining the osteotropism of tumour cells, which would eventually lead to bone colonisation. This could likely provide the answer to a quite intriguing question, that is why some cancers, such as prostate and breast, have a specific predilection to metastasise to the bone. Therefore, it is important to completely address the molecular mechanisms underlying this aspect of bone oncology, identifying relevant pathways, the targeting of which could make any type of bone metastasis curable or avoidable.
... Regarding the mechanisms underlying this interaction, several studies already documented an aberrant RANKL expression in different B cell malignancies such as CLL, multiple myeloma and follicular lymphoma [13][14][15][16][17][18][19][20][21] . Actually, Secchiero et al. reported that RANK/RANKL interaction potently increases the release of IL-8 as a factor able to promote clone proliferation and resistance to apoptotic signals in CLL 14 . ...
Skeletal erosion has been found to represent an independent prognostic indicator in patients with advanced stages of chronic lymphocytic leukaemia (CLL). Whether this phenomenon also occurs in early CLL phases and its underlying mechanisms have yet to be fully elucidated. In this study, we prospectively enrolled 36 consecutive treatment-naïve patients to analyse skeletal structure and bone marrow distribution using a computational approach to PET/CT images. This evaluation was combined with the analysis of RANK/RANKL loop activation in the leukemic clone, given recent reports on its role in CLL progression. Bone erosion was particularly evident in long bone shafts, progressively increased from Binet stage A to Binet stage C, and was correlated with both local expansion of metabolically active bone marrow documented by FDG uptake and with the number of RANKL + cells present in the circulating blood. In immune-deficient NOD/Shi-scid, γcnull (NSG) mice, administration of CLL cells caused an appreciable compact bone erosion that was prevented by Denosumab. CLL cell proliferation in vitro correlated with RANK expression and was impaired by Denosumab-mediated disruption of the RANK/RANKL loop. This study suggests an interaction between CLL cells and stromal elements able to simultaneously impair bone structure and increase proliferating potential of leukemic clone.
... RANK and RANKL are also implicated in the development of select lymphoid organs: gene-targeted mice deficient for RANK or RANKL lack peripheral lymph nodes, display thymic abnormalities, and are osteopetrotic with resultant reduction in B-cell development; in addition, a role for RANK and RANKL in the embryological development of Aire positive medullary thymic epithelial cells (mTEC) has been described (8,9,(45)(46)(47). RANKL blockade as a mechanism of altering E, Mean proportion of CD8 þ T-cellexpressing zero, one, two, or three cytokines (of IFNg, IL-2, and TNFa) from two pooled experiments is shown for each of four treatment groups as indicated. ...
Purpose: Novel partners for established immune checkpoint inhibitors in the treatment of cancer are needed to address the problems of primary and acquired resistance. The efficacy of combination RANKL and CTLA4 blockade in anti-tumor immunity has been suggested by recent case reports in melanoma. Here we provide a rationale for this combination in mouse models of cancer.
Experimental Design: The efficacy and mechanism of a combination of RANKL and CTLA4 blockade was examined by tumor infiltrating lymphocyte analysis, tumor growth and metastasis using a variety of neutralizing antibodies and gene-targeted mice.
Results: RANKL blockade improved the efficacy of anti-CTLA4 mAbs against solid tumors and experimental metastases, with regulatory T cell (Treg) depleting anti-CTLA4 mAbs of the mouse IgG2a isotype showing greatest combinatorial activity. The optimal combination depended on the presence of activating Fc receptors and lymphocytes (NK cells for metastatic disease and predominantly CD8⁺ T cells for subcutaneous tumor control), while anti-RANKL alone did not require FcR. The significantly higher T-cell infiltration into solid tumors post anti-RANKL and anti-CTLA-4 was accompanied by increased T-cell effector function (cytokine polyfunctionality), and anti-RANKL activity occurred independently of Treg depletion. The majority of RANKL expression in tumors was on T cells whereas RANK-expressing cells were mostly tumor-associated macrophages (TAMs), with some expression also observed on dendritic cells (DC) and myeloid derived suppressor cells (MDSCs).
Conclusions: These results provide a rationale for the further investigation of RANKL-RANK interactions in tumor immunity and a basis for development of translational markers of interest in human clinical trials.
... RANKL binds to the transmembrane receptor RANK on osteoclast precursors and initiates osteoclastogenesis [61,62]. It has been demonstrated that RANKL and RANK are required for osteoclastogenesis since transgenic rankl −/− and rank −/− mice had no osteoclasts or developed osteosclerotic bones [63,64]. Osteoprotegrin (OPG) is a soluble glycoprotein that negatively regulates osteoclastogenesis through sequestering RANKL, resulting in the block of its interaction with RANK [65]. ...
Prostate cancer tissue is composed of both cancer cells and host cells. The milieu of host components that compose the tumor is termed the tumor microenvironment (TME). Host cells can be those derived from the tissue in which the tumor originates (e.g., fibroblasts and endothelial cells) or those recruited, through chemotactic or other factors, to the tumor (e.g., circulating immune cells). Some immune cells are key players in the TME and represent a large proportion of non-tumor cells found within the tumor. Immune cells can have both anti-tumor and pro-tumor activity. In addition, crosstalk between prostate cancer cells and immune cells affects immune cell functions. In this review, we focus on immune cells and cytokines that contribute to tumor progression. We discuss T-regulatory and T helper 17 cells and macrophages as key modulators in prostate cancer progression. In addition, we discuss the roles of interleukin-6 and receptor activator of nuclear factor kappa-B ligand in modulating prostate cancer progression. This review highlights the concept that immune cells and cytokines offer a potentially promising target for prostate cancer therapy.
... In particular, these cytokines dysregulate the receptor activator of nuclear factor B ligand (RANKL) osteoprotegerin (OPG) system: normally RANKL is a strong activator of osteoclastogenesis, while OPG is linked with osteoblastogenesis; thus the interaction of RANK on the surface of osteoclasts with its ligand RANKL induces osteoclastogenesis and conversely the interaction with the osteoblast derived soluble decoy receptor; osteoprotegerin (OPG) blocks RANK interaction inhibiting osteoclasts formation [58,59]. In experimental models, proinflammatory cytokines induce RANKL and promote bone reabsorption with consecutive bone loss; moreover, activated T cells can directly trigger osteoclastogenesis through RANKL leading to bone [60]. ...
Malnutrition is a major complication of inflammatory bowel disease (IBD). This mini review is focusing on main determinants of malnutrition in IBD, the most important components of malnutrition, including lean mass loss and sarcopenia, as an emerging problem. Each one of these components needs to be well considered in a correct nutritional evaluation of an IBD patient in order to build a correct multidisciplinary approach. The review is then focusing on possible instrumental and clinical armamentarium for the nutritional evaluation.
... TNFRSF11B (tumor necrosis factor receptor superfamily member 11b) encodes a protein belonging to the TNF-receptor superfamily. Studies in a mouse counterpart also suggested that this protein and its ligand play a role in lymph node organogenesis and vascular calcification 44,45 . A cis-eQTL of TNFRSF11B was also identified in the present study, and its transcription levels were highly correlated with WBC (r = 0.329, p = 1.85 × 10 −8 ). ...
The liver is the central metabolic organ and exhibits fundamental functions in haematological traits. Hepatic expression, haematological, plasma biochemical, and body composition traits were assessed in a porcine model (n = 297) to establish tissue-specific genetic variations that influence the function of immune-metabolism-correlated expression networks. At FDR (false discovery rate) <1%, more than 3,600 transcripts were jointly correlated (r = |0.22–0.48|) with the traits. Functional enrichment analysis demonstrated common links of metabolic and immune traits. To understand how immune and metabolic traits are affected via genetic regulation of gene expression, eQTLs were assessed. 20517 significant (FDR < 5%) eQTLs for 1401 transcripts were identified, among which 443 transcripts were associated with at least one of the examined traits and had cis-eQTL (such as ACO1 (6.52 × 10⁻⁷) and SOD1 (6.41 × 10⁻³⁰). The present study establishes a comprehensive view of hepatic gene activity which links together metabolic and immune traits in a porcine model for medical research.
... Ingar Olsen et al. periodontal inflammatory infiltrate can lead to robustly increased osteoclast frequency and activity (osteoclastogenesis) associated with active bone destruction. RANKL melds bone resorptive events with activated immune cell-mediated RANKL expression (23,24). It is the key intermediary linking immune cells to pathogenic bone loss in both rheumatoid arthritis (25) and periodontal disease (26,27). ...
Porphyromonas gingivalis, a keystone pathogen in chronic periodontitis, has been found to associate with remote body organ inflammatory pathologies, including atherosclerosis and Alzheimer’s disease (AD). Although P. gingivalis has a plethora of virulence factors, much of its pathogenicity is surprisingly related to the overall immunosuppression of the host. This review focuses on P. gingivalis aiding suppression of the host’s adaptive immune system involving manipulation of cellular immunological responses, specifically T cells and B cells in periodontitis and related conditions. In periodontitis, this bacterium inhibits the synthesis of IL-2 and increases humoral responses. This reduces the inflammatory responses related to T- and B-cell activation, and subsequent IFN-γ secretion by a subset of T cells. The T cells further suppress upregulation of programmed cell death-1 (PD-1)-receptor on CD+cells and its ligand PD-L1 on CD11b+-subset of T cells. IL-2 downregulates genes regulated by immune response and induces a cytokine pattern in which the Th17 lineage is favored, thereby modulating the Th17/T-regulatory cell (Treg) imbalance. The suppression of IFN-γ-stimulated release of interferon-inducible protein-10 (IP-10) chemokine ligands [ITAC (CXCL11) and Mig (CXCL9)] by P. gingivalis capsular serotypes triggers distinct T cell responses and contributes to local immune evasion by release of its outer membrane vesicles. In atherosclerosis, P. gingivalis reduces Tregs, transforms growth factor beta-1 (TGFβ-1), and causes imbalance in the Th17 lineage of the Treg population. In AD, P. gingivalis may affect the blood–brain barrier permeability and inhibit local IFN-γ response by preventing entry of immune cells into the brain. The scarcity of adaptive immune cells in AD neuropathology implies P. gingivalis infection of the brain likely causing impaired clearance of insoluble amyloid and inducing immunosuppression. By the effective manipulation of the armory of adaptive immune suppression through a plethora of virulence factors, P. gingivalis may act as a keystone organism in periodontitis and in related systemic diseases and other remote body inflammatory pathologies.
... associations between genetic markers and ACPA-positive RA, suggesting a different genetic background that could explain the difference between outcomes involving ACPA-positive or ACPA-negative RA. 4 5 The balance between osteoblast and osteoclast activity is disturbed in systemic or local conditions that affect the skeleton, such as osteoporosis or RA. 6 The activity of these cells is mediated by the receptor activator of nuclear factor κ B (RANK)/receptor activator of nuclear factor κ B ligand (RANKL)/osteoprotegerin (OPG) system. Since the genes encoding these proteins are highly implicated in erosion pathogenesis, numerous studies have examined the potential implications of certain single nucleotide polymorphisms (SNPs) located on these genes and RA risk or presence of erosions. ...
Objectives
The RANK/RANKL/osteoprotegerin (OPG) system plays a central role in the pathogenesis of bone erosions in rheumatoid arthritis (RA). The aim of this study was to test the association between 11 single-nucleotide polymorphisms (SNPs) located on RANK, RANKL and OPG genes and anticitrullinated peptide antibody (ACPA) presence or erosions in RA.
Methods
Patients: This work was performed on three independent samples of French patients with RA: the Etude de Suivi des PolyArthrites Indifférenciées Récentes (ESPOIR) (n=632), Rangueil Midi-Pyrénées (RMP) (n=249) and French Rheumatoid Arthritis Genetic Consortium (FRAGC) (n=590) cohorts. Genotyping: the genotyping of 11 SNPs located on RANK, RANKL and OPG were performed by PCR. Statistical analyses: The association between the genotypes with ACPA or erosions was first tested in the ESPOIR cohort using a χ² test and, in the case of significant association, replicated in the RMP and FRACG cohorts. A meta-analysis on the three cohorts was performed using the Mantel-Haenszel method.
Results
One SNP on RANK (rs8086340) and three SNPs on RANKL (rs7984870, rs7325635, rs1054016) were significantly associated with ACPA presence, while one SNP on OPG (rs2073618) and one SNP on RANKL (rs7325635) were significantly associated with erosions in the ESPOIR cohort. Following meta-analysis performed on the three samples, the SNP on RANK and the GGG haplotype of the three SNPs located on RANKL were both significantly associated with ACPA presence, while only the SNP on OPG remained significantly associated with erosions.
Conclusions
This study identified one SNP located on RANK, one haplotype on RANKL associated with ACPA presence, and one SNP located on OPG associated with erosions in three different samples of French patients with RA.
... In addition, osteocytes secrete FGF23, a systemic hormone acting on kidneys to regulate phosphate excretion. The molecular mechanism underlying "immune-mediated" osteoclastogenesis was not elucidated until the discovery of RANKL, and by the evidence that RANKL is expressed not only in osteoblasts and bone marrow stromal cells, but also in T cells and B cells [26]. The involvement of RANKL expressed by T cells in pathogenic bone loss was first demonstrated in rheumatoid arthritis in rats. ...
Osteoimmunology is a rapidly evolving field of medical science that reveals the relationship between the immune system and bone metabolism. Receptor activator of nuclear factor kappa B ligand (RANKL), its receptor, receptor activator of nuclear factor kappaB (RANK), and a soluble decoy receptor for RANKL, osteoprotegerin (OPG), are three key molecules involved in this system. Understanding osteoimmunology will be central for orthodontic tooth movement related bone metabolism, and for the development of new means to prevent and control pathologic bone loss in diseases such as rheumatoid arthritis, periodontitis etc. This article focuses on the role of Osteoimmunology and its signaling mechanisms in physiologic and pathologic models of bone loss.
... [30] Osteoclasts are multinucleated giant cells with bone-resorbing capacity. [31][32][33] RANKL, IL-20, and macrophage colony-stimulating factor are essential for osteoclast formation, promoting the differentiation of osteoclast precursors into mature osteoclasts. ...
Background: Bone metastasis (BM) is common in patients with lung cancer. Osteolysis is caused by increased osteoclast activity. Interleukin-20 (IL-20) and receptor activator of nuclear factor kappa-B ligand (RANKL) are crucial for osteoclast formation. Osteoprotegerin (OPG) inhibits a receptor activator of RANKL/RANK signaling. The aims of this study were to analyze the serum levels of IL-20, OPG, and RANKL in patients with and without BM and to observe the effect of chemotherapy on these cytokines.
Patients and Methods: A total 54 cases of pathologically confirmed lung adenocarcinoma (ADC) and 18 healthy individuals (Control) were enrolled in this study. Eligible patients were divided into three groups (18 patients per group): ADC without BM (ADC), ADC with BM (ADC + BM), and ADC with BM treated with chemotherapy (ADC + BM + Chemo). Serum IL-20, RANKL, and OPG levels were analyzed by enzyme-linked immunosorbent assay.
Results: Serum IL-20, RANKL, and OPG levels in ADC + BM patients were significantly elevated compared with that in the Control or ADC groups (both P < 0.001). The serum cytokine levels were significantly lower following chemotherapy compared with that in patients who did not receive chemotherapy (P < 0.001).
Conclusions: Serum IL-20, RANKL, and OPG levels increase in patients with lung cancer and BMs. Chemotherapy suppresses the elevation of these cytokines.
Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals—cytokines, chemokines, growth factors, transcription factors, and morphogens—are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL’s involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL’s role in thymic normal physiology and disease processes.
The tumor microenvironment (TME) is a complex infrastructure composed of stromal, epithelial, and immune cells embedded in a vasculature ECM. The microenvironment surrounding mammary epithelium plays a critical role during the development and differentiation of the mammary gland, enabling the coordination of the complex multihormones and growth factor signaling processes. Progesterone/progesterone receptor paracrine signaling interactions in the microenvironment play vital roles in stem/progenitor cell function during normal breast development. In breast cancer, the female sex hormones, estrogen and progesterone, and growth factor signals are altered in the TME. Progesterone signaling modulates not only breast tumors but also the breast TME, leading to the activation of a series of cross-communications that are implicated in the genesis of breast cancers. This chapter reviews the evidence that progesterone and PR signaling modulates not only breast epitheliums but also the breast TME. Furthermore, crosstalk between estrogen and progesterone signaling affecting different cell types within the TME is discussed. A better understanding of how PR and progesterone affect the TME of breast cancer may lead to novel drugs or a therapeutic approach for the treatment of breast cancer shortly.
Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various signals, some of which are received directly from noncellular elements of the bone. In vitro, adherence to the bone triggers the reduction of cell–cell fusion events between osteoclasts and the activation of osteoclasts to form unusual dynamic cytoskeletal and membrane structures that are required for degrading the bone. Integrins on the surface of osteoclasts are known to receive regulatory signals from the bone matrix. Regulation of the availability of these signals is accomplished by enzymatic alterations of the bone matrix by protease activity and phosphorylation/dephosphorylation events. Other membrane receptors are present in osteoclasts and may interact with as yet unidentified signals in the bone. Bone mineral has been shown to have regulatory effects on osteoclasts, and osteoclast activity is also directly modulated by mechanical stress. As understanding of how osteoclasts and other bone cells interact with the bone has emerged, increasingly sophisticated efforts have been made to create bone biomimetics that reproduce both the structural properties of the bone and the bone’s ability to regulate osteoclasts and other bone cells. A more complete understanding of the interactions between osteoclasts and the bone may lead to new strategies for the treatment of bone diseases and the production of bone biomimetics to repair defects.
Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa. RANKL was associated with epithelial–mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Therefore, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa.
Bone provides structure and support for the body, protecting organs and providing levers for movement, but it is also an important metabolic organ. Osteocytes within mineralized bone regulate bone formation and resorption and also act as endocrine cells, producing fibroblast growth factor 23 (FGF23) in response to phosphate and 1,25(OH)2vitamin D. Diseases of FGF23 excess highlight the role of phosphate wasting on bone demineralization in the context of FGF23 excess. However, in vitro studies suggest that FGF23, alone and in conjunction with its cofactor Klotho, may also have direct effects on bone biology. The development of neutralizing antibodies to FGF23 opens the door for new options in the treatment of diseases of hypophosphatemic rickets; these antibodies also offer new opportunities to evaluate the direct and indirect effects of FGF23 on bone.
Autophagy plays a critical role in the maintenance of bone homeostasis. Osteoprotegerin (OPG) is an inhibitor of osteoclast-mediated bone resorption. However, whether autophagy is involved in the antiosteoclastogenic effects of OPG remains unclear. The present study aimed to investigate the potential mechanism of autophagy during OPG-induced bone resorption via inhibition of osteoclasts differentiated from bone marrow-derived macrophages in BALB/c mice. The results showed that after treatment with receptor activator of nuclear factor-κΒ ligand and macrophage colony-stimulating factor for 3 days, TRAP+ osteoclasts formed, representing the resting state of autophagy. These osteoclasts were treated with OPG and underwent autophagy, as demonstrated by LC3-II accumulation, acidic vesicular organelle formation, and the presence of autophagosomes. The levels of autophagy-related proteins, LC3-II increased and P62 decreased at 3 hr in OPG-treated osteoclasts. The viability, differentiation, and bone resorption activity of osteoclasts declined after OPG treatment. Treatment with OPG and chloroquine, an autophagy inhibitor, attenuated OPG-induced inhibition of osteoclastic bone resorption, whereas rapamycin (RAP), an autophagy inducer, enhanced OPG-induced inhibition of differentiation, survival, and bone resorption activity of osteoclasts. Furthermore, OPG reduced the amount of phosphorylated(p) protein kinase B (AKT) and pmTOR and increased the level of pULK, in a dose-dependant manner. LY294002, a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT pathway inhibitor, attenuated the decline in pAKT, but enhanced the decline in pmTOR and the increase in pULK1 following OPG treatment. RAP enhanced the OPG-induced increase in pULK1. The PI3K inhibitor 3-methyladenine partly blocked OPG-induced autophagy. Thus, the results revealed that OPG inhibits osteoclast bone resorption by inducing autophagy via the AKT/mTOR/ULK1 signaling pathway.
Background:
The aim of this study was to simultaneously and quantitatively assess the expression levels of 20 periodontal disease-related proteins in gingival crevicular fluid (GCF) from normal controls (NOR) and severe periodontitis (SP) patients with an antibody array.
Methods:
Antibodies against 20 periodontal disease-related proteins were spotted onto a glass slide to create a periodontal disease antibody array (PDD). The array was then incubated with GCF samples collected from 25 NOR and 25 SP patients. Differentially expressed proteins between NOR and SP patients were then used to build receiver operator characteristic (ROC) curves and compare five classification models, including Support Vector Machine, Random Forest, k Nearest Neighbor, Linear Discriminant Analysis, and Classification and Regression Trees.
Results:
Seven proteins (CRP, IL-1α, IL-1β, IL-8, MMP-13, Osteoprotegerin, Osteoactivin) were significantly upregulated in SP patients compared with NOR, while RANK was significantly downregulated. The highest diagnostic accuracy using a ROC curve was observed for IL-1β with an area under the curve of 0.984. Five of the proteins (IL-1β, IL-8, MMP-13, Osteoprotegerin, Osteoactivin) were identified as important features for classification. Linear Discriminant Analysis had the highest classification accuracy across the five classification models that were tested.
Conclusion:
This study highlights the potential of antibody arrays to diagnose periodontal disease. This article is protected by copyright. All rights reserved.
The activity and the distribution of osteogenic cells around octacalcium phosphate (OCP) granules combined with gelatin matrix (OCP/Gel) onlaid on rat calvaria were analyzed histomorphometrically and immunohistochemically during vertical bone augmentation under mechanical or non-mechanical environment until 8 weeks. OCP/Gel disk was placed in subperiosteal pocket on the calvaria with or without polytetrafluoroethylene (PTFE) support. The latter is a non-mechanical stress model to alleviate the mechanical stress from the subcutaneous tissues. Onlay grafts of gelatin (Gel) sponge disk and OCP granules were also carried out for the comparison purpose. When bone augmentation was evaluated in 1st area from bone surface (area until 150 μm high from bone surface) and 2nd area above the newly formed bone (area until 150 μm high from the 1st area), bone formation was enhanced most in 1st area followed by 2nd area of OCP/Gel with PTFE. The appearance of TRAP-positive osteoclast-like cells was suppressed more in the newly formed tissue with PTFE than those without PTFE with an emphasis of the presence of gelatin. Although Runx2 positive-cells were accumulated more in both OCP/Gel with and without PTFE, osteocalcin-positive cells were abundant in OCP/Gel with PTFE than that without PTFE, suggesting that non-mechanical stress condition is more suitable for osteoblast differentiation. The appearance of RANKL-positive cells was restrained in OCP/Gel and Gel with PTFE while OPG-positive cells were most accumulated in OCP/Gel without PTFE. The results suggest that OCP/Gel composite under the mechanical stress-alleviated condition can enhance bone augmentation through balanced osteoblastic and osteoclastic cellular activities. This article is protected by copyright. All rights reserved.
Denosumab is a human monoclonal antibody against RANKL. This antibody decreases bone turnover markers and increases bone mineral density (BMD) in postmenopausal women. In phase 3 studies including more than 1100 women, denosumab achieved greater increases in lumbar spine, total hip, distal 1/3 radius, and total BMD than alendronate 70 mg weekly. Recent data suggest that denosumab also decreases vertebral and non-vertebral fractures. This drug seems to be safe, although the most frequent side effects are arthralgia, back pain, and nasopharyngitis. No increased incidence of neoplasia has been found compared to placebo or alendronate. However, infections requiring inpatient treatment were more frequent in study groups treated with denosumab. These were common community acquired infections and were treated with standard antibiotics. No opportunistic infections were reported. Denosumab is a very promising new drug for the treatment of osteopenia and osteoporosis, and hopefully more long-term safety information and further fracture data will support its commercial use in the near future.
Aspirin (acetylsalicylic acid, ASA) has been shown to improve bone marrow mesenchymal stem cell-based calvarial bone regeneration by promoting osteogenesis and inhibiting osteoclastogenesis. However, it remains unknown whether aspirin influences other immune cells during bone formation. In the present study, we investigated whether ASA treatment influenced macrophage activation during the LPS inducement. We found that ASA could downregulate the expressions of iNOS and TNF-α both in mouse peritoneum macrophages and RAW264.7 cells induced by LPS via the IκK/IκB/NF-κB pathway and a COX2/PGE2/EP2/NF-κB feedback loop, without affecting the expressions of FIZZ/YM-1/ARG1 induced by IL-4. Furthermore, we created a rat mandibular bone defect model and showed that ASA treatment improved bone regeneration by inhibiting LPS-induced macrophage activation in the early stages of inflammation. Taken together, our results indicated that ASA treatment was a feasible strategy for improving bone regeneration, particularly in inflammatory conditions.
Oncogenic events combined with a favourable environment are the two main factors in the oncological process. The tumour microenvironment is composed of a complex, interconnected network of protagonists, including soluble factors suh as cytokines, extracellular matrix components, interacting with fibroblasts, endothelial cells, immune cells and various specific cell types depending on the location of the cancer cells (e.g. pulmonary epithelium, osteoblasts). This diversity defines specific "niches" (e.g. vascular, immune, bone niches) involved in tumour growth and the metastatic process. These actors communicate together by direct intercellular communications and/or in an autocrine/paracrine/endocrine manner involving cytokines and growth factors. Among these glycoproteins, RANKL and its receptor RANK, members of the TNF and TNFR superfamilies, have stimulated the interest of the scientific community. RANK is frequently expressed by cancer cells in contrast to RANKL which is frequently detected in the tumour microenvironment and together they participate in every step in cancer development. Their activities are markedly regulated by OPG (a soluble decoy receptor) and its ligands, and by LGR4, a membrane receptor able to bind RANKL. The aim of the present review is to provide an overview of the functional implication of the RANK/RANKL system in cancer development, and to underline the most recent clinical studies.
With the widespread use of glucocorticoids for systemic diseases affecting nearly every bodily system, exogenous glucocorticoid is an important cause of osteoporosis. This chapter highlights major aspects of advances as well as current perspectives on treatment options of glucocorticoid bone disease. The adverse effect of glucocorticoid therapy on bone mineral density (BMD) is not linear with time. Bone loss and fracture risk seems to be related, at least to some extent, to the dose and duration of glucocorticoid exposure. The risk of fracture increases rapidly after the commencement of glucocorticoid therapy, but decreases again after cessation of therapy. Glucocorticoids contribute to increased fracture risk through a number of direct and indirect mechanisms that result in accelerated bone loss. The bone loss is biphasic, precipitous during the first 12 months, and more gradual but continuous in subsequent years as measured by DXA or histomorphometry. There are several issues to consider in reviewing studies on the efficacy of therapies available for glucocorticoidinduced osteoporosis. These include primary versus secondary prevention, underlying disease states, and fracture prevention. A distinction needs to be made between primary and secondary prevention trials. The former commence at or soon after glucocorticoid therapy is begun, because the greatest rate of bone loss due to glucocorticoids occurs within the first 6–12 months of use. Secondary prevention, or treatment trials are those that commence after the subject has already taken glucocorticoids for a significant period of time and in whom bone loss has already occurred. Very few prospective studies have been undertaken in children to determine the long-term effects on bone mass of chronic glucocorticoid administration. However, as the large majority of peak bone mass is acquired during the first two decades of life, glucocorticoid exposure during this critical time of bone mass accrual may impact negatively on peak bone mass.
Crohn's disease and ulcerative colitis are associated with an increased risk of osteoporosis and bone fractures. Initialstudies suggested very high rates of osteoporosis in IBD based on bone mineral density (BMD) measurements;however, more recent studies suggest that BMD is often normal in IBD and average short-term changes are small.Doctors managing patients with IBD will have to consider a variety of risk factors and not just BMD measurements inassessing fracture risk. The evolution of knowledge regarding receptor for activated factor of nuclear factor kappa B (RANK), its ligand RANKLand osteoprotegerin (OPG) that serves as a decoy receptor, has enhanced our understanding of osteoporosis and alsoT cell immunobiology. Recent clinical studies in subjects with IBD have revealed that serum OPG levels may be elevated and inflamedintestinal tissue secretes OPG. It is suspected that OPG levels are elevated as a counter-regulatory response to lowBMD as serum OPG levels in IBD have been found to be inversely associated with BMD. OPG may ultimately prove tobe a useful therapeutic target for managing both low bone mass and colitis in patients with IBD. Currently, an underpinning of therapy for all IBD patients should include supplemental calcium and vitamin D,particularly for patients using corticosteroids. Postmenopausal women and men over age 50 years should be considered for BMD screening (in conjunction with anassessment of other risk factors for fracture) and if this suggests high fracture risk then pharmacological interventionmay be appropriate. Those with spontaneous or low-impact fractures that are typical for osteoporotic fractures (e.g.,hip fractures, one or more moderate to severe vertebral compression fractures) should be considered to haveestablished disease with high risk for further fractures and also require intervention.
In the bone marrow, hemopoietic stem cells proliferate and differentiate into lineage-restricted progenitors that eventually give rise to the terminally differentiated cells of the lymphoid and myeloid series. This differentiation process is directed by a group of lineage-specific growth factors, the colonystimulating factors (CSFs). CSF-1 is the lineage-specific growth factor for cells of the mononuclear phagocyte system (MNPS) and for osteoclasts. The cytokine binds to a single class of high-affinity cell surface receptors that are encoded by the proto-oncogene c-. fms and belong to the receptor tyrosine kinase family. This chapter discusses the effects of the cytokine on the cells of this lineage and explains the involved mechanisms. The analysis of the interactions of signaling pathways in osteoclasts has allowed for a dissection of the various effects exerted by the secreted and membrane-bound forms of CSF-1 on the respective target cells. CSF-1 is an essential growth factor for osteoclast-lineage cells. CSF-1 and RANKL are the only growth factors that under physiological conditions are indispensable for functional osteoclastogenesis. Deficiency in these factors or their receptors leads to osteopetrosis due to the absence of osteoclasts. CSF-1 is also one of the factors stimulating the accompanying bone resorption and contributes to the development of a suitable microenvironment for the tumors.
A major and recent advance of skeletal biology has been the realization that bone is an endocrine organ. One hormone secreted by the osteoblasts, the bone-forming cells, osteocalcin affects glucose homeostasis, energy expenditure and male fertility. This chapter will review the particularities of the mechanism of activation of osteocalcin, its mode of action and what it reveals for our understanding of the regulation of glucose homeostasis and male fertility. We will also present how the identification of a specific receptor for osteocalcin has allowed to verify that it has the same function in humans as in the mouse.
Lymphotoxin α (LTα)–deficient mice revealed critical roles for LTα in lymphoid organogenesis, but it is not clear whether LTα functions through an LTα homotrimer (LTα3) or LTα/β heterotrimers. We generated LTβ-deficient mice and found them to lack Peyer's patches, peripheral lymph nodes, splenic germinal centers, and follicular dendritic cells. Unlike LTα-deficient mice, LTβ-deficient mice had cervical and mesenteric lymph nodes. Furthermore, the mesenteric lymph nodes had germinal center–like regions, although these structures appeared to lack follicular dendritic cells. The absence of cervical and mesenteric lymph nodes in LTα-deficient mice, and yet their presence in LTβ-deficient mice and in mice deficient in tumor necrosis factor receptor types I and II, suggest that LTα3 may signal via an as yet unidentified receptor.
Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like
cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing
factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation.
Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin
(OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting
the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library
of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family
and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular
domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished
the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing
factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating
an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to
TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only
osteoclastogenesis but also immune system.
The nfkbl and nfkb2 genes encode closely related products regulating immune and inflammatory responses1−3. Their role during development and differentiation remains unclear. The generation of nfkb1 null mice (p50-/-) resulted in altered immune responses, but had no effect on development4. Similarly, nfkb2 knockout mice (p52-/-) did not show developmental defects (J.C. et al., manuscript submitted). We have investigated the potential for in vivo compensatory functions of these genes by generating double-knockout mice. The surprising result was that the animals developed osteopetrosis because of a defect in osteoclast differentiation, suggesting redundant functions of NF-B1 and NF-B2 proteins in the development of this cell lineage. The osteopetrotic phenotype was rescued by bone marrow transplantation, indicating that the hematopoietic component was impaired. These results define a new mouse osteopetrotic mutant and implicate NF-B proteins in bone development, raising new directions in the treatment of bone disorders.
Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation. In this study the physiological role of OPG is investigated by generating OPG-deficient mice. Adolescent and adult OPG-/- mice exhibit a decrease in total bone density characterized by severe trabecular and cortical bone porosity, marked thinning of the parietal bones of the skull, and a high incidence of fractures. These findings demonstrate that OPG is a critical regulator of postnatal bone mass. Unexpectedly, OPG-deficient mice also exhibit medial calcification of the aorta and renal arteries, suggesting that regulation of OPG, its signaling pathway, or its ligand(s) may play a role in the long observed association between osteoporosis and vascular calcification.
In mice deficient in either lymphotoxin-alpha (LT-alpha) or the type I tumor necrosis factor (TNF) receptor, but not the type II TNF receptor, germinal centers failed to develop in peripheral lymphoid organs. Germinal center formation was restored in LT-alpha-deficient mice by transplantation of normal bone marrow, indicating that the LT-alpha-expressing cells required to establish this lymphoid structure are derived from bone marrow.
To investigate the role of TNF alpha in the development of in vivo immune response we have generated TNF alpha-deficient mice by gene targeting. Homozygous mutant mice are viable and fertile, develop lymph nodes and Peyer's patches and show no apparent phenotypic abnormalities, indicating that TNF alpha is not required for normal mouse development. In the absence of TNF alpha mice readily succumb to L. monocytogenes infections and show reduced contact hypersensitivity responses. Furthermore, TNF alpha knockout mice are resistant to the systemic toxicity of LPS upon D-galactosamine sensitization, yet they remain sensitive to high doses of LPS alone. Most interestingly, TNF alpha knockout mice completely lack splenic primary B cell follicles and cannot form organized follicular dendritic cell (FDC) networks and germinal centers. However, despite the absence of B cell follicles, Ig class-switching can still occur, yet deregulated humoral immune responses against either thymus-dependent (TD) or thymus-independent (TI) antigens are observed. Complementation of TNF alpha functioning by the expression of either human or murine TNF alpha transgenes is sufficient to reconstitute these defects, establishing a physiological role for TNF alpha in regulating the development and organization of splenic follicular architecture and in the maturation of the humoral immune response.
We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) stimulate the differentiation of human monocytes into two phenotypically distinct types of macrophages. However, in vivo, not only CSF but also many other cytokines are produced under various conditions. Those cytokines may modulate the differentiation of monocytes by CSFs. In the present study, we showed that CD14+ adherent human monocytes can differentiate into CD1+relB+ dendritic cells (DC) by the combination of GM-CSF plus interleukin-4 (IL-4) and that they differentiate into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated giant cells (MGC) by the combination of M-CSF plus IL-4. However, the monocyte-derived DC were not terminally differentiated cells; they could still convert to macrophages in response to M-CSF. Tumor necrosis factor-alpha (TNF-alpha) stimulated the terminal differentiation of the DC by downregulating the expression of the M-CSF receptor, cfms mRNA, and aborting the potential to convert to macrophages. In contrast to IL-4, interferon-gamma (IFN-gamma) had no demonstrable effect on the differentiation of monocytes. Rather, IFN-gamma antagonized the effect of IL-4 and suppressed the DC and MGC formation induced by GM-CSF + IL-4 and M-CSF + IL-4, respectively. Taken together, these results provide a new aspect to our knowledge of monocyte differentiation and provide evidence that human monocytes are flexible in their differentiation potential and are precursors not only of macrophages but also of CD1+relB+DC and TRAP-positive MGC. Such a diverse pathway of monocyte differentiation may constitute one of the basic mechanisms of immune regulation.
A novel secreted glycoprotein that regulates bone resorption has been identified. The protein, termed Osteoprotegerin (OPG), is a novel member of the TNF receptor superfamily. In vivo, hepatic expression of OPG in transgenic mice results in a profound yet nonlethal osteopetrosis, coincident with a decrease in later stages of osteoclast differentiation. These same effects are observed upon administration of recombinant OPG into normal mice. In vitro, osteoclast differentiation from precursor cells is blocked in a dose-dependent manner by recombinant OPG. Furthermore, OPG blocks ovariectomy-associated bone loss in rats. These data show that OPG can act as a soluble factor in the regulation of bone mass and imply a utility for OPG in the treatment of osteoporosis associated with increased osteoclast activity.
Osteopetrotic (op/op) mice lack functional M-CSF and have depressed levels of macrophages and osteoclasts. We prepared transgenic mice (hMRP8bcl-2) that express human Bcl-2 in monocytes. In vitro hMRP8bcl-2 monocytes do not undergo apoptosis in the absence of serum and M-CSF, while op/op and wild-type monocytes die. These Bcl-2-expressing monocytes spontaneously undergo macrophage differentiation. In vivo, the op/op hMRP8bcl-2 mice show significant replenishment of tissue macrophages. Their long bone osteopetrosis is largely reversed, and extensive medullary hematopoiesis appears in the bone marrow. We propose that M-CSF augments monocyte survival, permitting them to respond to internal and external cues for their differentiation.
The tumor necrosis factor (TNF) family cytokines lymphotoxin (LT) alpha and LTbeta form heterotrimers that are expressed on the surface of activated lymphocytes and natural killer cells; LTalpha homotrimers can be secreted as well. Mice with a disrupted LTalpha gene lack lymph nodes (LN), Peyer's patches (PP), and follicular dendritic cell (FDC) networks and reveal profound defects of the splenic architecture. However, it is unclear which of these abnormalities is the result of the absence in LTalpha homotrimers or LTalphabeta heterotrimers. To distinguish between these two possibilities, a mouse strain deficient in LTbeta was created employing Cre/loxP-mediated gene targeting. Mice deficient in LTbeta reveal severe defects in organogenesis of the lymphoid system similar to those of LTalpha-/- mice, except that mesenteric and cervical LN are present in most LTbeta-deficient mice. Both LTbeta- and LTalpha-deficient mice show significant lymphocytosis in the circulation and peritoneal cavity and lymphocytic infiltrations in lungs and liver. After immunization, PNA-positive B cell clusters were detected in the splenic white pulp of LTbeta-deficient mice, but FDC networks were severely underdeveloped. Collectively, these results indicate that LTalpha can signal independently from LTbeta in the formation of PNA-positive foci in the spleen, and especially in the development of mesenteric and cervical LN.
An early stage in thymocyte development, after rearrangement of the β chain genes of the T cell receptor (TCR), involves expression
of the pre-TCR complex and accompanying differentiation of CD4−CD8− double negative (DN) cells to CD4+CD8+ double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated
motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70
or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes
undergo β chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells
after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping
functions in signaling from the pre-TCR and hence in early thymocyte development.
A novel member of the tumor necrosis factor (TNF) cytokine family, designated TRANCE, was cloned during a search for apoptosis-regulatory
genes using a somatic cell genetic approach in T cell hybridomas. The TRANCE gene encodes a type II membrane protein of 316 amino acids with a predicted molecular mass of 35 kDa. Its extracellular domain
is most closely related to TRAIL, FasL, and TNF. TRANCE is an immediate early gene up-regulated by TCR stimulation and is controlled by calcineurin-regulated transcription factors.
TRANCE is most highly expressed in thymus and lymph nodes but not in nonlymphoid tissues and is abundantly expressed in T
cells but not in B cells. Cross-hybridization of the mouse cDNA to a human thymus library yielded the human homolog, which
encodes a protein 83% identical to the mouse ectodomain. HumanTRANCE was mapped to chromosome 13q14 while mouseTRANCE was located to the portion of mouse chromosome 14 syntenic with human chromosome 13q14. A recombinant soluble form of TRANCE
composed of the entire ectodomain induced c-Jun N-terminal kinase (JNK) activation in T cells but not in splenic B cells or
in bone marrow-derived dendritic cells. These results suggest a role for this TNF-related ligand in the regulation of the
T cell-dependent immune response.
The T cell repertoire is shaped by positive and negative selection of thymocytes that express low levels of T cell receptor (TCR) and both CD4 and CD8. TCR-mediated signals that determine these selection processes are only partly understood. Vav, a GDP-GTP exchange factor for Rho-family proteins, is tyrosine phosphorylated following TCR stimulation, suggesting that it may transduce TCR signals. We now demonstrate that mice lacking Vav are viable and display a profound defect in the positive selection of both class I- and class II-restricted T cells. In contrast, Vav is not essential for negative selection, though in its absence negative selection is much less effective. Vav may influence the efficiency of TCR-induced selection events by regulating the intracellular calcium flux of thymocytes.
TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine) is a new member of the TNF family that is induced upon T cell receptor engagement and activates c-Jun N-terminal kinase (JNK) after interaction with its putative receptor (TRANCE-R). In addition, TRANCE expression is restricted to lymphoid organs and T cells. Here, we show that high levels of TRANCE-R are detected on mature dendritic cells (DCs) but not on freshly isolated B cells, T cells, or macrophages. Signaling by TRANCE-R appears to be dependent on TNF receptor-associated factor 2 (TRAF2), since JNK induction is impaired in cells from transgenic mice overexpressing a dominant negative TRAF2 protein. TRANCE inhibits apoptosis of mouse bone marrow-derived DCs and human monocyte-derived DCs in vitro. The resulting increase in DC survival is accompanied by a proportional increase in DC-mediated T cell proliferation in a mixed leukocyte reaction. TRANCE upregulates Bcl-xL expression, suggesting a potential mechanism for enhanced DC survival. TRANCE does not induce the proliferation of or increase the survival of T or B cells. Therefore, TRANCE is a new DC-restricted survival factor that mediates T cell-DC communication and may provide a tool to selectively enhance DC activity.
NF-kappaB is a family of related, dimeric transcription factors that are readily activated in cells by signals associated with stress or pathogens. These factors are critical to host defense, as demonstrated previously with mice deficient in individual subunits of NF-kappaB. We have generated mice deficient in both the p50 and p52 subunits of NF-kappaB to reveal critical functions that may be shared by these two highly homologous proteins. We now demonstrate that unlike the respective single knockout mice, the p50/p52 double knockout mice fail to generate mature osteoclasts and B cells, apparently because of defects that track with these lineages in adoptive transfer experiments. Furthermore, these mice present markedly impaired thymic and splenic architectures and impaired macrophage functions. The blocks in osteoclast and B-cell maturation were unexpected. Lack of mature osteoclasts caused severe osteopetrosis, a family of diseases characterized by impaired osteoclastic bone resorption. These findings now establish critical roles for NF-kappaB in development and expand its repertoire of roles in the physiology of differentiated hematopoietic cells.
The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.
4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.
CT1 is a carbohydrate moiety of CD45 that is expressed on fetal thymocytes in vivo. Examination of CT1 expression on thymocyte subsets revealed that primarily pro-T cells (CD44+ CD25+) and pre-T cells (CD44- CD25+) expressed CT1. Interestingly, non-T-lineage committed lymphoid progenitors (CD44+ CD25-) lacked CT1 indicating temporal regulation of expression of this determinant in early T-lineage committed development. In addition, CT1 was expressed by the majority of thymocytes in RAG-2(-/-) mice where thymocyte development is blocked at the CD44- CD25+ stage. Since late pre-T cells (CD44- CD25-) lacked the CT1 epitope we tested whether pre-TCR triggering regulated CT1 expression. Injection of CD3epsilon-specific mAb into RAG-2(-/-) mice induces differentiation of immature thymocytes to the double-positive stage of thymocyte development. Using this system, we demonstrated that expression of CT1 by RAG-2(-/-) thymocytes was rapidly lost from pre-T cells following anti-CD3 mAb treatment. Furthermore, the decline in CT1 expression induced by CD3 signaling paralleled a loss of mRNA for the glycosyltransferase responsible for the addition of CT1 to CD45. Flow cytometric analysis also revealed that the loss of the CT1 epitope was inversely correlated with an increase in peanut agglutinin ligand expression, demonstrating a complex regulation of cell surface glycosylation at a critical juncture in thymocyte development.
The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.
We investigated lymphotoxin (LT) and TNF function in lymph node genesis and cellular organization by manipulating LTβ-R and TNF-R signaling. Lymph nodes developed in LTα−/− mice treated in utero with agonist anti-LTβ-R monoclonal antibody. Thus, LTβ-R signaling mediates lymph node genesis. Surprisingly, mucosal lymph nodes that can develop independently of LTαβ/LTβ-R interaction were generated. Normal mice treated in utero with LTβ-R-Ig and TNF-R55-Ig or anti-TNF lacked all lymph nodes, indicating that TNF signaling contributes to lymph node genesis. Lymph nodes generated in LTα−/− mice had disrupted cellular organization. Therefore, LTβ-R signaling during gestation is not sufficient to establish normal cellular microarchitecture. We conclude that LT and TNF play critical roles in the genesis and cellular organization of lymph nodes.
Immature thymocytes express a pre–T cell receptor (pre-TCR) composed of the TCRβ chain paired with pre-Tα. Signals from this
receptor are essential for passage of thymocytes through a key developmental checkpoint in the thymus. These signals were
efficiently delivered in vivo by a truncated form of the murine pre-TCR that lacked all of its extracellular immunoglobulin
domains. De novo expression of the truncated pre-TCR or an intact αβTCR was sufficient to activate characteristic TCR signaling
pathways in a T cell line. These findings support the view that recognition of an extracellular ligand is not required for
pre-TCR function.
A new soluble mediator was found in supernatant fluid from cultures of human peripheral blood leukocytes that were stimulated
by phytohemagglutinin, or by antigenic material present in human dental plaque deposits. This soluble Jactor produced bone
resorption in organ cultures of fetal rat bones as measured by increased release of calcium-45, and also increased the number
of active osteoclasts.
In T-cell precursors, the T-cell-receptor beta chain is expressed before the T-cell-receptor alpha chain and is sufficient to advance T-cell development in the absence of T-cell receptor alpha chains. In immature T cells, the T-cell-receptor beta protein can form disulphide-linked heterodimers with the pre-T-cell-receptor alpha chain and associate with signal-transducing CD3 molecules. The recently cloned pre-T-cell-receptor alpha gene encodes a transmembrane protein that is expressed in immature but not mature T cells. Here we show that alpha beta, but not gamma delta, cell development is severely hampered in pre-T-cell-receptor alpha-gene-deficient mice, which establishes a crucial role for the pre-T-cell receptor in early thymocyte development.
Mice lacking the proto-oncogene c-fos develop the bone disease osteopetrosis. Fos mutant mice were found to have a block in
the differentiation of bone-resorbing osteoclasts that was intrinsic to hematopoietic cells. Bone marrow transplantation rescued
the osteopetrosis, and ectopic c-fos expression overcame this differentiation block. The lack of Fos also caused a lineage
shift between osteoclasts and macrophages that resulted in increased numbers of bone marrow macrophages. These results identify
Fos as a key regulator of osteoclast-macrophage lineage determination in vivo and provide insights into the molecular mechanisms
underlying metabolic bone diseases.
We describe the spontaneous development of inflammatory bowel disease (IBD) in several immunodeficient mouse strains created via gene targeting in embryonic stem cells. Chronic colitis was observed in T cell receptor (TCR) alpha mutant, TCR beta mutant, TCR beta x delta double mutant, or class II major histocompatibility complex (MHC) mutant mice, but not in recombination-activating gene RAG-1 mutant mice or nude mice kept in the same specific pathogen-free animal facility. This clinical pattern suggests that the disease requires the presence of B lymphocytes and the absence of class II MHC-restricted CD4+ alpha beta T cells. IBD in the mutant mice has some of the features of the human disease ulcerative colitis. Based on these results, we suggest that dysfunction of the mucosal immune system may underly the pathogenesis of some types of IBD in humans.
Experimental auction markets were used to investigate the acceptability to undergraduate students of milk from cows treated with bST. The auction market was used to elicit willingness of participants to pay to exchange "bST milk" for "normal" milk when given repeated market participation. The results suggest that more than 60% of participants would purchase milk from cows treated with bST at economically feasible discounts.
Mice rendered deficient in lymphotoxin (LT) by gene targeting in embryonic stem cells have no morphologically detectable lymph
nodes or Peyer's patches, although development of the thymus appears normal. Within the white pulp of the spleen, there is
failure of normal segregation of B and T cells. Spleen and peripheral blood contain CD4+CD8- and CD4-CD8+ T cells in a normal
ratio, and both T cells subsets have an apparently normal lytic function. Lymphocytes positive for immunoglobulin M are present
in increased numbers in both the spleen and peripheral blood. These data suggest an essential role for LT in the normal development
of peripheral lymphoid organs.
Bone-resorbing osteoclasts are of hemopoietic cell origin, probably of the CFU-M-derived monocyte-macrophage family. Bone marrow-derived osteoblastic stromal cells play an important role in modulating the differentiation of osteoclast progenitors in two different ways: one is the production of soluble factors, and the other is cell-to-cell recognition between osteoclast progenitors and osteoblastic stromal cells. M-CSF is probably the most important soluble factor, which appears to be necessary for not only proliferation of osteoclast progenitors, but also differentiation into mature osteoclasts and their survival. A number of local factors as well as systemic hormones induce osteoclast differentiation. They are classified into three categories in terms of the signal transduction: vitamin D receptor-mediated signals [1 alpha,25(OH)2D3]; protein kinase A-mediated signals (PTH, PTHrP, PGE2, and IL-1); and gp130-mediated signals (IL-6, IL-11, oncostatin M, and leukemia inhibitory factor). All of these osteoclast-inducing factors appear to act on osteoblastic cells to commonly induce osteoclast differentiation factor (ODF), which recognizes osteoclast progenitors and prepares them to differentiate into mature osteoclasts. This line of approach will undoubtedly produce new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment such as osteoporosis, osteopetrosis, Paget's disease, rheumatoid arthritis, and periodontal disease.
Felix R, Hofstetter W, Cecchini MG. Recent developments in the understanding of the pathophysiology of osteopetrosis. Eur J Endocrinol 1996;134:143–56. ISSN 0804–4643
Osteopetrosis is a rare metabolic bone disease characterized by a generalized increase in skeletal mass. It is inherited in a number of mammalian species, including man, and results from a congenital defect in the development or function of the osteoclasts. The consequent impairment of bone resorption prevents formation of bone marrow cavities, causes delayed or absent tooth eruption and results often in abnormally shaped bone. The pathogenetic defect may be intrinsic either to the osteoclast lineage or to the mesenchymal cells that constitute the microenvironment supporting the development and activation of the osteoclasts. In the first example, the disease can be cured by transplantation of hemopoietic cells. In some cases, bone marrow transplantation has also been successful in curing human osteopetrosis. This, together with the variability in the age of onset and severity of clinical aspects, suggests that a multiplicity of genetic mutations may cause the human disease. In recent years the genetic effects of some osteopetrotic mutations have been identified. This new information has been essential for the understanding of osteoclast biology. Colony stimulating factor 1 (CSF-1), the growth factor for cells of the mononuclear phagocytic system, is also essential for the development of osteoclasts. In the osteopetrotic ( op ) mouse, no biologically active CSF-1 is synthesized due to a point mutation in the coding region of its gene. This leads to an almost complete lack of osteoclast development and to impaired bone resorption. Altered CSF-1 production seems also to be involved in the toothless ( tl ) rat osteopetrosis. Recently, the mutation responsible for the microphthalmic ( mi ) mouse osteopetrosis has been identified in the gene encoding a member of the basic-helix-loop-helixleucine zipper (bHLH-ZIP) protein family of transcription factors. The mi gene product seems to play a role in the fusion process of osteoclast precursor cells. Finally, osteopetrosis has been the result of experimental gene disruption in mice. Targeted disruption of the c-src proto-oncogene encoding a non-receptor tyrosine kinase leads to a form of osteopetrosis where osteoclasts are present but inactive. This indicates that pp60c− src , localized primarily on ruffled border membranes and vacuoles of the osteoclasts, is important for osteoclastic function. Disruption of the c-fos proto-oncogene, a major component of the AP-1 transcription factor complex, leads to an osteopetrotic phenotype characterized by a complete absence of osteoclasts. The defect is intrinsic to hemopoietic precursors that are unable to progress beyond an early stage of osteoclast differentiation. In humans, deficiency of carbonic anhydrase II has been identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. A lack of expression of the vacuolar proton pump has been observed in osteoclasts of a patient with craniometaphyseal dysplasia. In conclusion, the disease, although rare, is of great pathophysiological relevance for our understanding of the processes that govern the development and function of osteoclasts.
R Felix, Department of Pathophysiology, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland
Interactions between stromal cells and thymocytes play a crucial role in T cell development. The thymic stroma is complex and consists of epithelial cells derived from the pharyngeal region during development, together with macrophages and dendritic cells of bone marrow origin. In addition, fibroblasts and matrix molecules permeate the whole framework. It is now apparent that these individual stromal components play specialized roles at different stages of T cell differentiation. Thus, at the early CD4 ⁻ 8 ⁻ stage of development, T cell precursors require fibroblast as well as epithelial cell interactions. Later, at the CD4 ⁺ 8 ⁺ stage, as well as providing low avidity TCR/MHC-peptide interactions, thymic epithelial cells have been shown to possess unique properties essential for positive selection. Dendritic cells, on the other hand, are probably efficient mediators of negative selection, but they may not be solely responsible for this activity.
Alongside the functional roles of stromal cells, considerable progress is being made in unraveling the nature of the signaling pathways involved in T cell development. Identification of the pre-T cell receptor (pre-TCR) and associated signaling molecules marks an important advance in understanding the mechanisms that control gene rearrangement and allelic exclusion. In addition, a better understanding of the signaling pathways that lead to positive selection on the one hand and negative selection on the other is beginning to emerge.
Many issues remain unresolved, and some are discussed in this review. What, for example, is the nature of the chemotactic factor(s) that attract stem cells to the thymus? What is the molecular basis of the essential interactions between early thymocytes and fibroblasts, and early thymocytes and epithelial cells? What is special about cortical epithelial cells in supporting positive selection? These and other issues are ripe for analysis and can now be approached using a combination of modern molecular and cellular techniques.
While early thymic T cell precursor populations and their maturational sequence have been recently identified, the signals driving differentiation are unknown. While cytokines may play an integral role in T cell development, various mouse models rendered genetically deficient for specific cytokines do not display abnormalities in T cell development. Recently, we have generated IL-7 -/- mice and reported that IL-7 plays a unique and nonredundant role in lymphopoiesis. These mice displayed a 10- to 20-fold reduction in the total number of T and B cells. Here, we show that IL-7 -/- mice display a sharp reduction in both the frequency and absolute number of adult thymic gamma delta T cells while retaining normal frequencies of alpha beta T cells. This defect in gamma delta T cell production extends to peripheral organs as IL-7 -/- mice are essentially devoid of splenic and intestinal intraepithelial gamma delta T cells. This aberrant phenotype was traced back to impaired fetal gamma delta T cell maturation. In the absence of IL-7, differentiation of immature V gamma 3 low-CD24+ fetal T cells to mature V gamma 3 high CD24- cells is inhibited. In contrast, NK cell maturation appears to be only mildly affected in the absence of IL-7. To further clarify the role of IL-7 in thymic development, detailed analysis of CD3-4-8- thymic precursors was performed. A partial inhibition in the differentiation of CD44+25+ pro-T cells into CD44-25+ pre-T cells was observed. Unexpectedly, the lack of IL-7 resulted in decreased expression of CD117 (c-kit) on both CD4 low and pro-T cells, suggesting that IL-7 may influence the expression of other cytokine receptors involved in early hemopoietic development. Together, these data clarify the developmental abnormalities during T cell development due to the absence of IL-7.
Much has been learned about the cell biology and molecular biology of the osteoclast in the last 5 yr. The osteoclast appears to be derived from CFU-GM, the committed monocyte-granulocyte precursor cell. This cell then differentiates into more committed precursors for the osteoclast. The role of the marrow microenvironment appears to be critical for murine osteoclast formation, although in human systems it appears to be nonessential but acts to enhance osteoclast formation and resorption. The osteoclast has been shown to be a secretory cell capable of producing both stimulators and inhibitors of osteoclast formation and resorption. The identification of the role of protooncogenes, such as c-fos and pp60c-src, in osteoclast differentiation and bone resorption has provided important insights into the regulation of normal osteoclast activity. Studies such as these should help us to dissect the pathophysiology of abnormal osteoclastic activity, such as seen in hypercalcemia of malignancy, osteopetrosis, and Paget's disease of bone. Future research is needed to further delineate the signaling pathways involved in osteoclastic bone resorption in response to cytokines and hormones, as well as to identify the molecular events required for commitment of multipotent precursors to the osteoclast lineage. Development of osteoclast cell lines may be possible and would greatly enhance our understanding of the biology of the osteoclast. Utilization of current model systems to examine the effects of cytokines and hormones on osteoclast precursors in vitro and in vivo and the ability to obtain large numbers of highly purified osteoclasts for production of osteoclast cDNA libraries should lead to important new discoveries in osteoclast biology.
The pre-T cell receptor (pre-TCR) that minimally consists of the TCR beta chain and the disulfide-linked pre-T cell receptor alpha (pT alpha) chain in association with signal-transducing CD3 molecules rescues from programmed cell death cells with productive TCR beta rearrangements. The pre-TCR induces expansion and differentiation of these cells such that they become TCR alpha beta bearing CD4+8+ thymocytes, which express only a single TCR beta chain and then either die of neglect or--upon TCR-ligand interaction--undergo either positive or negative selection. The newly discovered pT alpha gene encodes a transmembrane protein that belongs to the Ig superfamily and contains a cytoplasmic tail that, however, has no essential function in signal transduction, which is mediated by CD3 molecules and most likely p56lck. Experiments in pT alpha gene-deficient mice show that the pre-TCR has a crucial role in maturation as well as allelic exclusion of alpha beta T cells but is not required for the development of gamma delta-expressing cells. The function of the pre-TCR cannot be fully assumed by an alpha beta TCR that is expressed abnormally early in T cell development.
Osteoclasts are terminally differentiated multinucleated cells that are the principal resorptive cells of bone, playing a central role in the formation of the skeleton and regulation of its mass. The molecular events involved in the differentiation and function of osteoclasts had not been clarified for a long time. Over the past two decades, several novel approaches have been developed and adopted to investigate osteoclast biology. In the present review, we would like to update recent progress in the elucidation of the molecular mechanism of osteoclast activation and function.
For a brief period during fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules surprisingly express the Peyer's patch addressin MAdCAM-1. This expression allows initial seeding of this incipient structure by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. We show here that CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1 and that can become natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. Since the necessity of lymphotoxin in lymphoid organ development has been shown, we propose that the novel subset of CD4+CD3-LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.
The nfkb1 and nfkb2 genes encode closely related products regulating immune and inflammatory responses. Their role during development and differentiation remains unclear. The generation of nfkb1 null mice (p50-/-) resulted in altered immune responses, but had no effect on development. Similarly, nfkb2 knockout mice (p52-/-) did not show developmental defects (J.C. et al., manuscript submitted). We have investigated the potential for in vivo compensatory functions of these genes by generating double-knockout mice. The surprising result was that the animals developed osteopetrosis because of a defect in osteoclast differentiation, suggesting redundant functions of NF-kappaB1 and NF-kappaB2 proteins in the development of this cell lineage. The osteopetrotic phenotype was rescued by bone marrow transplantation, indicating that the hematopoietic component was impaired. These results define a new mouse osteopetrotic mutant and implicate NF-kappaB proteins in bone development, raising new directions in the treatment of bone disorders.
Dendritic cells are rare haematopoietic cells that reside in a number of organs and tissues. By capturing, processing and presenting antigens to T cells, dendritic cells are essential for immune surveillance and the regulation of specific immunity. Several members of the tumour necrosis factor receptor (TNFR) superfamily are integral to the regulation of the immune response. These structurally related proteins modulate cellular functions ranging from proliferation and differentiation to inflammation and cell survival or deaths. The functional activity of dendritic cells is greatly increased by signalling through the TNFR family member CD40. Here we report the characterization of RANK (for receptor activator of NF-kappaB), a new member of the TNFR family derived from dendritic cells, and the isolation of a RANK ligand (RANKL) by direct expression screening. RANKL augments the ability of dendritic cells to stimulate naive T-cell proliferation in a mixed lymphocyte reaction, and increases the survival of RANK+ T cells generated with interleukin-4 and transforming growth factor (TGF)-beta. Thus RANK and RANKL seem to be important regulators of interactions between T cells and dendritic cells.
The morphogenesis and remodeling of bone depends on the integrated activity of osteoblasts that form bone and osteoclasts that resorb bone. We previously reported the isolation of a new cytokine termed osteoclastogenesis inhibitory factor, OCIF, which specifically inhibits osteoclast development. Here we report the cloning of a complementary DNA of human OCIF. OCIF is identical to osteoprotegerin (OPG), a soluble member of the tumor-necrosis factor receptor family that inhibits osteoclastogenesis. Recombinant human OPG/OCIF specifically acts on bone tissues and increases bone mineral density and bone volume associated with a decrease of active osteoclast number in normal rats. Osteoblasts or bone marrow-derived stromal cells support osteoclastogenesis through cell-to-cell interactions. A single class of high affinity binding sites for OPG/OCIF appears on a mouse stromal cell line, ST2, in response to 1,25-dihydroxyvitamin D3. An anti-OPG/OCIF antibody that blocks the binding abolishes the biological activity of OPG/OCIF. When the sites are blocked with OPG/OCIF, ST2 cells fail to support osteoclastogenesis. These results suggest that the sites are involved in cell-to-cell signaling between stromal cells and osteoclast progenitors and that OPG/OCIF inhibits osteoclastogenesis by interrupting the signaling through the sites.
Osteoclasts, the main protagonists involved in bone resorption mechanisms, are generally considered to be of haematopoietic origin, although the exact nature of the primary osteoclastic stem cells is still unknown. In vitro cellular models developed to study the different events of osteoclastic differentiation have revealed that not only several cell types (osteoblasts, monocytes, lymphocytes, etc.) but also many soluble factors (cytokines, hormones, vitamins, ions, etc.) and extracellular matrix elements (osteopontin, osteocalcin, etc.) are involved in osteoclastic differentiation and activation. This article provides an exhaustive review of recent knowledge on the origin of the osteoclast and the main substances involved in the osteoclastogenesis and activation of these cells.
Osteoclast differentiation factor (ODF), a ligand for osteoprotegerin (OPG)/osteoclastogenesis-inhibitory factor (OCIF), induces osteoclast-like cell formation in vitro. To elucidate the role of ODF in the microenvironment of bone, we examined effects of ODF, OPG/OCIF, and anti-ODF polyclonal antibody on bone resorption using a fetal mouse long bone culture system. A genetically engineered soluble-form ODF (sODF) elicited bone resorption in a concentration-dependent manner and OPG/OCIF blocked the bone resorption. Anti-ODF polyclonal antibody, which neutralizes ODF activity, negated bone resorption induced by 1 alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2. OPG/OCIF also abolished bone-resorbing activity elicited by these bone-resorbing agents. Interleukin 1 alpha-stimulated bone resorption was also significantly suppressed by anti-ODF polyclonal antibody and OPG/OCIF. Thus, we conclude that ODF plays a critical role in bone resorption in the microenvironment of bone.
Lymphotoxin and tumor necrosis factor provide essential signals for the formation of secondary lymphoid tissue structures. Lymphotoxin in its membrane form (LT alpha 1 beta 2 heterotrimer) is required for the development of lymph nodes and Peyer's patches and supports the development of normal spleen structure. In the spleen, lymphotoxin acts during embryonic development to support the formation of distinct B and T cell zones. Lymphotoxin also acts in a tonic fashion-supporting the formation and maintenance of the follicular dendritic cell network and of primary B cell follicle structure. The cells that deliver the tonic lymphotoxin signal supporting follicular dendritic cell structure are B cells; thus, B cells participate fundamentally in the development of the lymphoid tissue structure in which they subsequently mature.
The recently cloned osteoclastogenesis inhibitory factor, or osteoprotegerin (OPG), has been shown to be a potent inhibitor of osteoclast formation. The inhibition is believed to be mediated through specific binding of OPG to a cell surface ligand on osteoblastic stromal cells. In this report we have studied the effect of the bone resorbing agent prostaglandin E2 (PGE2) on OPG mRNA levels in primary cultures of human bone marrow stroma cells (hBMSC). PGE2 dose- and time-dependently down-regulated the mRNA levels of OPG, as measured by RNAse protection assay. After 4 hours of stimulation with 1 microM PGE2, OPG mRNA levels were significantly decreased. The inhibitory effect was seen at and above 1 nM of PGE2. To elucidate whether the OPG mRNA levels are regulated via the proteinkinase A and/or the proteinkinase C pathways we stimulated cells with either forskolin (FSK) or phorbolic ester (PDbu) respectively. FSK (10 microM) decreased OPG mRNA levels to 50 % of control, whereas PE (10 nM) upregulated the mRNA levels to 250 % of control. These data show that PGE2 down-regulates the expression of OPG mRNA in hBMSC, probably via an increase in cAMP. This mechanism might be involved in PGE2-induced bone resorption.
Osteoclasts are multinucleated cells that resorb bone. Osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), acts as a naturally occurring decoy receptor for osteoclast differentiation factor, which mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts. Here we show that the OCIF/OPG knockout mice exhibited severe osteoporosis due to enhanced osteoclastogenesis when they grew to be adults. These mice were viable and fertile. They exhibited marked bone loss accompanied by destruction of growth plate and lack of trabecular bone in their femurs. The strength of their bones dramatically decreased. These results demonstrate that OCIF/OPG is a key factor acting as a negative regulator against osteoclastogenesis. The OCIF/OPG knockout mice provide the first animal model for osteoporosis without other obvious abnormalities.
The leukocyte-specific adapter molecule SLP-76 (Src homology 2 domain–containing leukocyte protein of 76 kilodaltons) is rapidly
phosphorylated on tyrosine residues after receptor ligation in several hematopoietically derived cell types. Mice made deficient
for SLP-76 expression contained no peripheral T cells as a result of an early block in thymopoiesis. Macrophage and natural
killer cell compartments were intact in SLP-76–deficient mice, despite SLP-76 expression in these lineages in wild-type mice.
Thus, the SLP-76 adapter protein is required for normal thymocyte development and plays a crucial role in translating signals
mediated by pre–T cell receptors into distal biochemical events.
We investigated lymphotoxin (LT) and TNF function in lymph node genesis and cellular organization by manipulating LTbeta-R and TNF-R signaling. Lymph nodes developed in LTalpha-/- mice treated in utero with agonist anti-LTbeta-R monoclonal antibody. Thus, LTbeta-R signaling mediates lymph node genesis. Surprisingly, mucosal lymph nodes that can develop independently of LTalphabeta/LTbeta-R interaction were generated. Normal mice treated in utero with LTbeta-R-Ig and TNF-R55-Ig or anti-TNF lacked all lymph nodes, indicating that TNF signaling contributes to lymph node genesis. Lymph nodes generated in LTalpha-/- mice had disrupted cellular organization. Therefore, LTbeta-R signaling during gestation is not sufficient to establish normal cellular microarchitecture. We conclude that LT and TNF play critical roles in the genesis and cellular organization of lymph nodes.
Lymphotoxin beta receptor (LTbetaR)-/- mice were created by gene targeting. LTbetaR-/- mice lacked Peyer's patches, colon-associated lymphoid tissues, and all lymph nodes. Mucosa patrolling alphaEbeta7high integrin+ T cells were virtually absent. Spleens lost marginal zones; T/B cell segregation and follicular dendritic cell networks were absent. Peanut agglutinin+ cells were aberrantly detectable around central arterioles. In contrast to TNF receptor p55-/- mice, antibody affinity maturation was impaired. Since LTbetaR-/- mice exhibit distinct defects when compared to LTalpha-/- and LTbeta-/- mice, it is suggested that the LTbetaR integrates signals from other TNF family members. Thus, the LTbetaR proves pivotal for the ontogeny of the secondary lymphoid tissues. Furthermore, affinity maturation is dependent on LTalpha1beta2 rather than on LTalpha3.
Two physiological regulators of osteoclast maturation have recently been identified: the secreted protein osteoprotegerin and the cell-surface ligand to which it binds. These proteins are likely to play an important part in the control of bone resorption, but are also likely to have important roles in other tissues.
Advances in bone biology: The osteoclast.
- Roodman GD
Roodman GD. Advances in bone biology: The osteoclast.
Endocr. Rev. 1996; 17: 308–32.
TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor
- B R Wong
- R Josien
- Lee Sy
Wong BR, Josien R, Lee SY et al. TRANCE (tumor necrosis
factor [TNF]-related activation-induced cytokine), a new TNF
family member predominantly expressed in T cells, is a dendritic
cell-specific survival factor. J. Exp. Med. 1997; 186: 2075-80.