RANKL In Osteoclastogenesis Research Articles

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL.

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

LOX Fails to Substitute for RANKL in Osteoclastogenesis.

Osteoclasts are the exclusive bone-resorbing cells that have a central role in bone homeostasis as well as bone destruction in cancer and autoimmune disease. Both mouse and human genetic studies have clearly proven that receptor activator of NF-κB ligand (RANKL; encoded by the Tnfsf11 gene) and its receptor RANK are essential for osteoclastogenesis. Although there have been several reports on RANKL-independent osteoclastogenesis, previous studies have never provided in vivo evidence showing RANKL can be substituted by other molecules using RANKL- or RANK-deficient genetic backgrounds. Thus, to date, there is no clear evidence of RANKL-independent osteoclastogenesis and no molecule has ever been proven capable of inducing osteoclast differentiation more efficiently than RANKL. Recently, lysyl oxidase (LOX), the enzyme that mediates collagen cross-linking, has been shown to induce human osteoclasts in the absence of RANKL and has a stronger osteoclastogenic activity than RANKL. Here, we investigated the effect of LOX on osteoclast differentiation using RANKL- and RANK-deficient cells to strictly explore RANKL-independent osteoclastogenesis. CD14+ human peripheral blood cells as well as osteoclast precursor cells derived from wild-type, RANKL- and RANK-deficient mice were treated with RANKL and/or LOX in short-term (3 days) or long-term (3 weeks) experimental settings. LOX treatment alone did not result in the formation of tartrate-resistant acid phosphatase (TRAP)+ cells or resorption pits in either short-term or long-term culture. In combination with RANKL, long-term treatment with LOX synergistically promoted osteoclastogenesis in cells derived from wild-type mice; however, this was abrogated in RANKL-deficient cells. Long-term treatment with LOX stimulated RANKL expression in mouse bone marrow stromal cells via the production of reactive oxygen species (ROS). Furthermore, LOX injection failed to rescue the phenotype of RANKL-deficient mice. These results suggest that LOX has the ability to induce RANKL expression on stromal cells; however, it fails to substitute for RANKL in osteoclastogenesis. © 2016 American Society for Bone and Mineral Research.

Design of a RANK-Mimetic Peptide Inhibitor of Osteoclastogenesis with Enhanced RANKL-Binding Affinity.

The receptor activator of nuclear factor κB (RANK) and its ligand RANKL are key regulators of osteoclastogenesis and well-recognized targets in developing treatments for bone disorders associated with excessive bone resorption, such as osteoporosis. Our previous work on the structure of the RANK-RANKL complex revealed that Loop3 of RANK, specifically the non-canonical disulfide bond at the tip, performs a crucial role in specific recognition of RANKL. It also demonstrated that peptide mimics of Loop3 were capable of interfering with the function of RANKL in osteoclastogenesis. Here, we reported the structure-based design of a smaller peptide with enhanced inhibitory efficiency. The kinetic analysis and osteoclast differentiation assay showed that in addition to the sharp turn induced by the disulfide bond, two consecutive arginine residues were also important for binding to RANKL and inhibiting osteoclastogenesis. Docking and molecular dynamics simulations proposed the binding mode of the peptide to the RANKL trimer, showing that the arginine residues provide electrostatic interactions with RANKL and contribute to stabilizing the complex. These findings provided useful information for the rational design of therapeutics for bone diseases associated with RANK/RANKL function.

Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model

Osteoclasts are macrophage-related bone resorbing cells of hematopoietic origin. Factors that regulate osteoclastogenesis are of great interest for investigating the pathology and treatment of bone diseases such as osteoporosis. In mammals, receptor activator of NF-κB ligand (Rankl) is a regulator of osteoclast formation and activation: its misexpression causes osteoclast stimulation and osteoporotic bone loss. Here, we report an osteoporotic phenotype that is induced by overexpression of Rankl in the medaka model. We generated transgenic medaka lines that express GFP under control of the cathepsin K promoter in osteoclasts starting at 12 days post-fertilization (dpf), or Rankl together with CFP under control of a bi-directional heat-shock promoter. Using long-term confocal time-lapse imaging of double and triple transgenic larvae, we monitored in vivo formation and activation of osteoclasts, as well as their interaction with osteoblasts. Upon Rankl induction, GFP-positive osteoclasts are first observed in the intervertebral regions and then quickly migrate to the surface of mineralized neural and haemal arches, as well as to the centra of the vertebral bodies. These osteoclasts are TRAP (tartrate-resistant acid phosphatase) and cathepsin K positive, mononuclear and highly mobile with dynamically extending protrusions. They are exclusively found in tight contact with mineralized matrix. Rankl-induced osteoclast formation resulted in severe degradation of the mineralized matrix in vertebral bodies and arches. In conclusion, our in vivo imaging approach confirms a conserved role of Rankl in osteoclastogenesis in teleost fish and provides new insight into the cellular interactions during bone resorption in an animal model that is useful for genetic and chemical screening.

Antioxidant α-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-κB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-κB ligand (RANKL). Tumor necrosis factor-α (TNF-α) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of α-lipoic acid (α-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, α-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-α (RANKL/TNF-α). α-LA abolished ROS elevation by RANKL or RANKL/TNF-α and inhibited NF-κB activation in osteoclast precursor cells. Specifically, α-LA reduced DNA binding of NF-κB but did not inhibit IKK activation. Furthermore, α-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-α in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-κB regulation and the antioxidant α-lipoic acid has a therapeutic potential for bone erosive diseases.

RANK signaling is not required for TNFalpha-mediated increase in CD11(hi) osteoclast precursors but is essential for mature osteoclast formation in TNFalpha-mediated inflammatory arthritis.

To address the controversy of whether TNFalpha can compensate for RANKL in osteoclastogenesis in vivo, we used a TNFalpha-induced animal model of inflammatory arthritis and blocked RANKL/RANK signaling. TNFalpha increased osteoclast precursors available for RANK-dependent osteoclastogenesis. RANK signaling is not required for the TNFalpha-stimulated increase in CD11b(hi) osteoclast precursors but is essential for mature osteoclast formation. Although critical roles of TNFalpha in inflammatory arthritis and RANKL in bone resorption have been firmly established, a central controversy remains about the extent to which TNFalpha can compensate for RANKL during osteoclastogenesis and the stage at which RANK signaling is required for osteoclastogenesis. Here, we used the human TNFalpha transgenic mouse model (TNF-Tg) of erosive arthritis to determine if there are both RANK-dependent and -independent stages of osteoclastogenesis in TNFalpha-induced erosive arthritis. Osteoclastogenesis and osteoclast precursor (OCP) frequency were analyzed using histology, fluorescence-activated cell sorting (FACS), and cell culture from (1) TNF-Tg mice treated with the RANKL antagonist, RANK:Fc, or (2) TNF-Tg X RANK -/- mice generated by crossing TNF-Tg mice with RANK-/- mice. Treatment of TNF-Tg mice, which have increased OCPs in their spleens, with RANK:Fc dramatically reduced osteoclast numbers on the surface of their arthritic joints and within their bones, but did not decrease CD11b(hi) OCP numbers in their spleens. Long-term RANK:Fc administration alleviated joint erosion. Furthermore, TNF-Tg x RANK -/- mice had severe osteopetrosis, no osteoclasts, and no joint erosion, but increased CD11b(hi) precursor numbers that failed to form mature osteoclasts in vitro. RANK signaling is essential for mature osteoclast formation in TNFalpha-mediated inflammatory arthritis but not for the TNFalpha-induced increase in CD11b(hi) OCP that subsequently can differentiate into osteoclasts in inflamed joints.