Academic literature on the topic 'RANKL mutants'

The purpose of the present study was to assess the early stages of development of mouse first molar roots in the osteopetrotic context of RANKL invalidation in order to demonstrate that the radicular phenotype observed resulted not only from defective osteoclasts, but also from loss of cell-to-cell communication among dental, periodontium and alveolar bone cells involving RANKL signaling. Two experimental models were used in this study: Rankl mutants with permanent RANKL invalidation, and C57BL/6J mice injected during the first postnatal week with a RANKL neutralizing antibody corresponding to a transient RANKL invalidation. The dento-alveolar complex was systematically analyzed using micro-CT, and histological and immunohistochemical approaches. These experiments showed that the root elongation alterations observed in the Rankl-/- mice were associated with reduced proliferation of the RANK-expressing HERS cells with a significant decrease in proliferating cell nuclear antigen (PCNA) expression and a significant increase in P21 expression. The phenotypic comparison of the adult first molar root at 35 days between permanent and transitory invalidations of RANKL made it possible to demonstrate that alterations in dental root development have at least two origins, one intrinsic and linked to proliferation/differentiation perturbations in dental-root-forming cells, the other extrinsic and corresponding to disturbances of bone cell differentiation/function.

Abstract Abstract 3039 Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NF-κB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin as key regulators of bone remodelling. Multiple myeloma (MM) disrupts the balance within this molecule system towards osteoclastogenesis and bone destruction. Neutralization of RANKL by the monoclonal antibody Denosumab (AMG162) is presently being evaluated for treatment of both non-malignant and malignant osteolysis. We found, in line with previously published data, that primary MM cells (9 of 10) express substantial levels of RANKL at the cell surface and that MM cells directly release RANKL in soluble form (sRANKL). Next we evaluated the possibility to combine neutralization of sRANKL with targeting of MM cells for antibody-dependent cellular cytotoxicity (ADCC) of NK cells utilizing RANK-Ig fusion proteins with modified Fc portions. Compared to wildtype RANK-Fc, our mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (RANK-Fc-ADCC) and abrogated (RANK-Fc-KO) affinity, respectively, to the NK cell FcγRIIIa, but comparable capacity to neutralize RANKL in binding competition and osteoclast formation assays. Analyses with RANKL transfectants and RANKL-negative controls confirmed the high and lacking potential of the RANK-Fc-ADCC and the RANK-Fc-KO to induce NK ADCC, respectively, and ascertained that the RANK-Fc-ADCC specifically induced NK cell lysis of RANKL-expressing but not RANKL-negative target cells. Most notably, in cultures of NK cells with RANKL-expressing primary MM cells RANK-Fc-ADCC potently enhanced NK cell degranulation, cytokine release and MM cells lysis due to enhanced NK reactivity. Thus, our Fc-engineered RANK-Fc-ADCC fusion protein may both neutralize detrimental effects of sRANKL and enhance NK anti-tumor reactivity by targeting RANKL-expressing malignant cells thereby constituting an attractive immunotherapeutic means for treatment of MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 411 Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NFκB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) as key regulators of bone resorption. Multiple myeloma (MM) disrupts the balance within this molecule system, and severe bone destruction due to inappropriate osteoclastogenesis is a prominent feature of this disease. Besides MM cells, other malignant hematopoietic cells have also been found to express RANKL at the cell surface and to release this molecule in soluble form (sRANKL). Neutralization of RANKL using RANK-Fc fusion protein or monoclonal antibody (Denosumab/AMG162), which mimics the RANKL-neutralizing endogenous effects of osteoprotegerin, decreases osteolysis in multiple in vivo models and is presently being evaluated as a means to treat both non-malignant and malignant osteolysis. We here confirmed and extended previously published data and report that all investigated MM cell lines (n = 5) as well as primary leukemic cells of CLL patients (n = 12) displayed substantial levels of RANKL mRNA and surface expression. Moreover, we report that substantial levels of sRANKL can be detected in culture supernatants of MM and primary CLL cells, but not in supernatants of healthy PBMC. Next we engineered RANK-Fc fusion proteins with modified affinity to FcγR by mutating amino acids in the Fc portion as previously described (Lazar et al., PNAS 2006; Armour et al., Eur J Immunol 1999). Compared to wildtype RANK-Fc (RANK-Fc-wt), our mutants (S239D/I332E and E233P/L234V/L235A/DeltaG236/A327G/A330S) displayed highly enhanced and abrogated (RANK-Fc-ADCC+ and RANK-Fc-KO, respectively) affinity to FcγRIIIa expressed on NK cells, which play an important role in anti-tumor immunity due to their ability to lyse target cells directly and to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of therapeutic antibodies. The RANK-Fc-ADCC+ displayed similar capacitiy to neutralize sRANKL compared to the RANK-Fc-KO and the RANK-Fc-wt as revealed by binding competition assays. Next we cultured NK cells with L cells or P815 cells transfected to express RANKL and the parental, RANKL-negative controls in the presence or absence of the different RANK-Fc constructs. Addition of RANK-Fc-KO or RANK-Fc-wt did not substantially alter NK cell reactivity against the target cells. However, presence of the RANK-Fc-ADCC+ dramatically enhanced NK cell cytotoxicity and cytokine production in cultures with the RANKL-expressing target cells (increase from 20% to 89%, E:T ratio 30:1 and 12 pg/ml to 290 pg/ml respectively; both p<0.01, Student's t-test). Neither of the three RANK-Fc proteins altered NK cell cytotoxicity and cytokine production in cultures with the RANKL negative controls demonstrating that the RANK-Fc-ADCC+ specifically induced NK cell reactivity against RANKL-expressing malignant cells. Moreover, treatment with RANK-Fc-ADCC+ also significantly augmented NK cell anti-tumor reactivity in cultures with RANKL-expressing primary CLL cells of patients, and this was observed both in settings using allogenic NK cells and analyzing autologous NK cells among PBMC of the leukemia patients (both p<0.01, Student's t-test). Taken together, our Fc-engineered RANK-Fc-ADCC+ fusion protein may neutralize detrimental effects of sRANKL, can target RANKL-expressing malignant cells for NK cell anti-tumor reactivity and may thus constitute an attractive immunotherapeutic means for treatment of hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the “master” protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1–dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligase-inactive TRAF6 mutants. The IL-1–induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKL-induced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity.

ABSTRACT β-Catenin-dependent canonical Wnt signaling plays an important role in bone metabolism by controlling differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. To investigate its function in osteocytes, the cell type constituting the majority of bone cells, we generated osteocyte-specific β-catenin-deficient mice (Ctnnb1 loxP/loxP ; Dmp1-Cre). Homozygous mutants were born at normal Mendelian frequency with no obvious morphological abnormalities or detectable differences in size or body weight, but bone mass accrual was strongly impaired due to early-onset, progressive bone loss in the appendicular and axial skeleton with mild growth retardation and premature lethality. Cancellous bone mass was almost completely absent, and cortical bone thickness was dramatically reduced. The low-bone-mass phenotype was associated with increased osteoclast number and activity, whereas osteoblast function and osteocyte density were normal. Cortical bone Wnt/β-catenin target gene expression was reduced, and of the known regulators of osteoclast differentiation, osteoprotegerin (OPG) expression was significantly downregulated in osteocyte bone fractions of mutant mice. Moreover, the OPG levels expressed by osteocytes were higher than or comparable to the levels expressed by osteoblasts during skeletal growth and at maturity, suggesting that the reduction in osteocytic OPG and the concomitant increase in osteocytic RANKL/OPG ratio contribute to the increased number of osteoclasts and resorption in osteocyte-specific β-catenin mutants. Together, these results reveal a crucial novel function for osteocyte β-catenin signaling in controlling bone homeostasis.

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.

Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser144, catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains.

Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.