Receptor Activator Of Nuclear kappa-B Ligand Research Articles

Targeting receptor-activator of nuclear kappaB ligand in aneurysmal bone cysts: verification of target and therapeutic response

Aneurysmal bone cyst (ABC) is a benign tumor of bone presenting as a cystic, expansile lesion in both the axial and appendicular skeleton. Axial lesions demand special consideration, because treatment-related morbidity can be devastating. In similar lesions, such as giant cell tumor of bone (GCTB), the receptor-activator of nuclear kappaB ligand (RANKL)-receptor-activator of nuclear kappaB (RANK) signaling axis is essential to tumor progression. Although ABC and GCTB are distinct entities, they both contain abundant multinucleated giant cells and are osteolytic characteristically. We hypothesize that ABCs express both RANKL and RANK similarly in a cell-type specific manner, and that targeted RANKL therapy will mitigate ABC tumor progression. Cellular expression of RANKL and RANK was determined in freshly harvested ABC samples using laser confocal microscopy. A consistent cell-type-specific pattern was observed: fibroblastlike stromal cells expressed RANKL strongly whereas monocyte/macrophage precursor and multinucleated giant cells expressed RANK. Relative RANKL expression was determined by quantitative real-time polymerase chain reaction in ABC and GCTB tissue samples; no difference in relative expression was observed (P > 0.05). In addition, we review the case of a 5-year-old boy with a large, aggressive sacral ABC. After 3 months of targeted RANKL inhibition with denosumab, magnetic resonance imaging demonstrated tumor shrinkage, bone reconstitution, and healing of a pathologic fracture. Ambulation, and bowel and bladder function were restored at 6 months. Denosumab treatment was well tolerated. Post hoc analysis demonstrated strong RANKL expression in the pretreatment tumor sample. These findings demonstrate that RANKL-RANK signal activation is essential to ABC tumor progression. RANKL-targeted therapy may be an effective alternative to surgery in select ABC presentations.

Abstract 1426: Functional analysis of membrane bound versus soluble RANKL: implications for cancer-induced osteolysis.

Abstract The primary site of prostate cancer metastasis is the skeleton with the resultant metastases containing areas of extensive osteogenesis and osteolysis. The latter is a process controlled by multinucleated osteoclasts and receptor activator of nuclear kappa B ligand (RANKL) is critical for osteoclastogenesis. RANKL is a type II transmembrane molecule that forms a trimer on the cell surface and mediates its effects via RANK. We have shown that matrix metalloproteinases-3 and -7 (MMP-3, MMP-7) can process RANKL to a soluble form, sRANKL. The objective of this study is to determine the functionality of membrane bound RANKL versus sRANKL in promoting osteoclastogenesis. Full-length murine RANKL cDNA was isolated and inserted into the pcDNA 3.1(+) expression construct. In vitro translation experiments demonstrated that RANKL was being generated and could be processed by MMP-3 and MMP-7. N-terminal amino acid sequencing revealed that the site of cleavage occurs in the juxtamembrane region of RANKL between amino acids 145 and 146. We hypothesized that cleavage in the juxtamembrane region could generate an active soluble form of sRANKL. Osteoclastogenesis and resorption assays with myeloid precursors demonstrated the functionality of sRANKL. To determine whether RANKL or sRANKL is the major mediator of osteoclastogenesis in vivo we have generated RANKL mutants that are resistant to cleavage by MMPs and other proteases. Using a site directed mutagenesis kit we have designed a number of mutants and found that Δ158, (deletion of amino acids 71-157) and, Δ148, deletion of amino acids 72-147), were resistant to proteolytic cleavage. We anticipate that the while Δ158 and Δ148 are resistant to cleavage, they will be functional. We will therefore test the functionality of these two deletion mutants in-vitro by co-culturing myeloid precursor cells with Cos-7/Cos-1 cells expressing wild type and mutant RANKL proteins. In conclusion, we have demonstrated that RANKL is proteolytically cleaved to generate a soluble active form. We have generated non-cleavable mutant forms of RANKL that will allow us to assess the role of membrane bound versus soluble RANKL in-vitro and in-vivo. The results of these experiments will further elucidate the roles of RANKL and sRANKL in prostate to bone metastases in addition to being informative for the engineering of future RANKL based therapies. Citation Format: Jeremy J. McGuire, Mitsuru Futakuchi, Conor Lynch. Functional analysis of membrane bound versus soluble RANKL: implications for cancer-induced osteolysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1426. doi:10.1158/1538-7445.AM2013-1426

Inhibition of JNK in synovium by treatment with golimumab in rheumatoid arthritis

The aim of this study was to investigate immunohistological changes in mitogen-activated protein kinases (MAPKs) in the synovium following treatment with golimumab, compared with methotrexate (MTX). We assessed synovial tissues for 13 different molecules to detect cytokine levels histologically from 10 methotrexate (MTX)-treated rheumatoid arthritis (RA) patients as controls and 10 golimumab plus MTX-treated RA patients. Synovium samples from both groups were assessed by hematoxylin and eosin (HE) staining and analyzed for expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), CD4 (T cells), CD8 (T cells), CD20 (B cells), CD68 (macrophages), receptor activator of nuclear (kappa) B ligand (RANKL), bromodeoxyuridine (BrdU), CD29 (β-1 integrin), phospho-p38 MAPK (Tyr180/Tyr182), phospho-p44/42 MAPK (ERK1/ERK2), and phospho-c-Jun N-terminal kinase (JNK), by an immunohistological examination. HE staining showed that there was a significant decrease in cell proliferation in the synovium in RA patients who received golimumab compared with the controls. TNF-α, IL-6, MMP3, BrdU, p38, and ERK were not seen at significant levels in either group. On the other hand, CD4, CD8, CD20, CD29, CD68, RANKL, and JNK were significantly decreased in the golimumab group compared with the control. Based on a histological analysis of the synovium, it appears that the efficacy of the treatment with golimumab may involve the inhibition of cell proliferation, with decreases in T cells, B cells, macrophages, β-1 integrin, RANKL, and JNK in the synovium, compared with MTX treatment, in RA.

Decrease of CD68 and MMP-3 expression in synovium by treatment of adalimumab for rheumatoid arthritis

In order to investigate the histological change in the secondary non-responder cases of adalimumab compared with methotrexate (MTX) treatment, we performed immunohistochemical examination of synovial tissue by seven different molecules to detect expression patterns of cytokines. We histologically assessed synovial tissues from five MTX-treated rheumatoid arthritis (RA) patients as controls and five adalimumab plus MTX-treated RA patients after arthroscopic synovectomy in the knee joints. The synovium of both groups were assessed by hematoxylin and eosin (H&E) and analyzed the positive expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), B-cell precursor and mature B-cell transmembrane protein, CD20, macrophage marker, CD68, vascular endothelial growth factor (VEGF), receptor activator of nuclear (kappa) B ligand (RANKL) by immunohistochemical examination. H&E staining showed the increase of vascular and cell proliferations in the synovium of the RA patients who received adalimumab compared with the controls. TNF-α, IL-6 and CD20 were not significantly different in either group. On the other hand, MMP-3 and CD68 showed a significant decrease in the adalimumab group compared with controls (P < 0.05). VEGF and RANKL were weakly positive in both groups. Based on the histological analysis of synovium, the effect attenuation of adalimumab may be involved in vascular and cell proliferations; however, there was inhibition of the expression of CD68 and MMP-3 in synovium. These findings indicate CD68 and MMP-3 may have key roles in the mechanism of efficacy of adalimumab.

Osteoprotegerin expression in bone marrow by treatment with tocilizumab in rheumatoid arthritis

The aim of this study was to investigate histological changes of bone marrow in response to tocilizumab for rheumatoid arthritis (RA). After tocilizumab therapy, bone marrow tissues were extracted from ten RA patients at the time of total knee arthroplasty (TKA). Control samples were obtained from ten RA patients who underwent MTX mono-therapy. Histological examination of structural differences between the tocilizumab and control groups in bone marrow was performed using hematoxylin and eosin (H&E) to evaluate differences. In immunohistochemical examination, the expression of seven molecules including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), CD68, osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL), CD4 and osteopontin (OPN) were compared between two groups. NTx was significantly low at 44.5 ± 2 nM BCE/mM Cr compared with control at 73.2 ± 8 nM BCE/mM Cr. Immunohistochemical examination revealed that the bone marrow tissues of the RA patients who underwent tocilizumab therapy demonstrated significant positive OPG as compared with the control. However, immunohistochemical examinations after tocilizumab revealed that TNF-α, IL-6, CD68, CD4, OPN and RANKL were not significantly different with control of MTX in bone marrow. Therefore, treatment with tocilizumab increased the expression of OPG as the histological changes with respect to inhibit RANKL-related bone resorption of bone marrow in RA.

Histological changes in bone marrow after treatment of infliximab for rheumatoid arthritis

To investigate histological evidence of bone remodeling in response to infliximab for rheumatoid arthritis (RA), bone marrow tissues were extracted from ten RA patients at the time of total knee arthroplasty after treatment of infliximab for an average of 16 months (range, 8-24 months). The patients had a mean age of 65.3 years (range, 57-76 years) with 4.8 mg/week of methotrexate (MTX; 4-6 mg) and 3.8 mg/day of prednisolone (2-5 mg). Control samples were obtained from ten RA patients who did not undergo infliximab therapy. These patients had an average age of 67.6 years (range, 59-78 years) and received 5.2 mg/week of MTX (4-6 mg) and 4.0 mg/day of prednisolone (2-5 mg). Histological examination of structural differences between the infliximab and control groups in bone marrow was performed using hematoxylin and eosin (H & E) to evaluate differences. In immunohistochemical examination, the expressions of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), receptor activator of nuclear (kappa) B ligand (RANKL), osteoprotegerin (OPG), and osteopontin (OPN) were compared between both groups. H & E staining revealed that the bone marrow tissues of the RA patients who underwent infliximab therapy demonstrated newly formed thickness of interstitial septum among the trabeculae as compared with the control group. Moreover, immunohistochemical examinations revealed that TNF-alpha, IL-6, RANKL, OPG, and OPN were expressed in this newly formed bone after infliximab therapy. Therefore, treatment with infliximab improved the histological changes with respect to bone metabolism in the newly formed bone marrow tissues.

Therapeutic agents for disorders of bone and calcium metabolism--Denosumab, a fully human monoclocal antibody-targeting RANKL as a therapy for cancer-induced bone diseases

Receptor activator of nuclear-kappaB ligand (RANKL) is a protein expressed by oseoblastic stromal cells, binds to receptor activator of nuclear factor-kappaB (RANK) and is the primary mediator of osteoclast differentiation, activation, and survival. RANKL is responsible for osteoclast-mediated bone resorption in a broad range of conditions and play a key role in establishment and propagation of skeletal disease in patients with advanced cancer. Denosumab is a fully human monoclonal antibody that binds RANKL with high affinity and specificity and inhibits RANKL-RANK interaction, mimicking the endogenous effects of osteoprotegerin, a soluble RANKL decoy receptor. In the phase 1 clinical trials in healthy post menopausal women and patients with multiple myeloma or breast cancer with bone metastasis including Japanese (except for multiple myeloma) showed that single and multiple subcutaneous injection of denosumab caused rapid and sustained suppression of markers of osteoclastic bone resorption with favorable safety profiles. Currently, the larger global clinical trials to investigate the effect of this agent for the treatment of cancer-induced bone disease as well as osteoporosis are underway.

Human cytomegalovirus, Epstein‐Barr virus and bone resorption‐inducing cytokines in periapical lesions of deciduous teeth

A connection of herpesvirus periapical infection with symptomatic and large-size periapical lesions has been recognized in adult patients, but no data exist about a possible involvement of herpesviruses in severe periapical pathosis in children. Herpesviruses have the potential to elicit potent bone resorption-inducing cytokines in mammalian cells. This study aimed to determine the occurrence of human cytomegalovirus and Epstein-Barr virus DNA, and mRNA transcripts of receptor activator of nuclear kappa B ligand (RANKL), osteoprotegerin, core binding factor alpha-1, colony stimulating factor-1, transforming growth factor-beta, and monocyte chemoattractant protein-1 in periapical symptomatic pathosis of deciduous teeth. Twelve deciduous molar teeth from patients aged 2-8 years were extracted due to severe periapical infection, and granulomatous tissue adherent to the root tip of the extracted teeth was collected using a surgical knife. Non-diseased pulpal tissue, obtained from 12 teeth extracted for orthodontic reasons, served as negative control. Polymerase chain reaction assays were employed to identify herpesvirus DNA and cytokine gene expression, using established polymerase chain reaction primers and procedures. Seven (58%) of the periapical lesions yielded human cytomegalovirus and eight (67%) Epstein-Barr virus. Only one (8%) periapical lesion showed neither human cytomegalovirus nor Epstein-Barr virus. In healthy pulpal tissue, one (8%) specimen demonstrated human cytomegalovirus and another (8%) specimen revealed Epstein-Barr virus. Of the cytokines examined, RANKL expression showed significantly higher occurrence in periapical pathosis than in healthy pulpal tissue (P < 0.040). No relationship was identified between the type of herpesvirus and cytokine expression in the periapical lesions studied. The present findings provide evidence of a putative role of human cytomegalovirus and Epstein-Barr virus in the pathogenesis of symptomatic periapical pathosis in deciduous teeth. Increased RANKL expression in periapical lesions may be of pathogenetic significance.

Mechanical Strain Differentially Regulates Endothelial Nitric-oxide Synthase and Receptor Activator of Nuclear κB Ligand Expression via ERK1/2 MAPK

Exercise promotes positive bone remodeling through controlling cellular processes in bone. Nitric oxide (NO), generated from endothelial nitric-oxide synthase (eNOS), prevents resorption, whereas receptor activator of nuclear kappa B ligand (RANKL) promotes resorption through regulating osteoclast activity. Here we show that mechanical strain differentially regulates eNOS and RANKL expression from osteoprogenitor stromal cells in a magnitude-dependent fashion. Strain (0.25-2%) induction of eNOS expression was magnitude-dependent, reaching a plateau at 218 +/- 36% of control eNOS. This was accompanied by increases in eNOS protein and a doubling of NO production. Concurrently, 0.25% strain inhibited RANKL expression with increasing response up to 1% strain (44 +/- 3% of control RANKL). These differential responses to mechanical input were blocked when an ERK1/2 inhibitor was present during strain application. Inhibition of NO generation did not prevent strain-activated ERK1/2. To confirm the role of ERK1/2, cells were treated with an adenovirus encoding a constitutively activated MEK; Ad.caMEK significantly increased eNOS expression and NO production by more than 4-fold and decreased RANKL expression by half. In contrast, inhibition of strain-activated c-Jun kinase failed to prevent strain effects on either eNOS or RANKL. Our data suggest that physiologic levels of mechanical strain utilize ERK1/2 kinase to coordinately regulate eNOS and RANKL in a manner leading to positive bone remodeling.