osteoprotegerin and Chondrocalcinosis

To study the mechanism by which the readthrough mutation in TNFRSF11B, encoding osteoprotegerin (OPG) with additional 19 amino acids at its C-terminus (OPG-XL), causes the characteristic bidirectional phenotype of subchondral bone turnover accompanied by cartilage mineralization in chondrocalcinosis patients.. OPG-XL was studied by human induced pluripotent stem cells expressing OPG-XL and two isogenic CRISPR/Cas9-corrected controls in cartilage and bone organoids. Osteoclastogenesis was studied with monocytes from OPG-XL carriers and matched healthy controls followed by gene expression characterization. Dual energy X-ray absorptiometry scans and MRI analyses were used to characterize the phenotype of carriers and non-carriers of the mutation.. Human OPG-XL carriers relative to sex- and age-matched controls showed, after an initial delay, large active osteoclasts with high number of nuclei. By employing hiPSCs expressing OPG-XL and isogenic CRISPR/Cas9-corrected controls to established cartilage and bone organoids, we demonstrated that expression of OPG-XL resulted in excessive fibrosis in cartilage and high mineralization in bone accompanied by marked downregulation of MGP, encoding matrix Gla protein, and upregulation of DIO2, encoding type 2 deiodinase, gene expression, respectively.. The readthrough mutation at CCAL1 locus in TNFRSF11B identifies an unknown role for OPG-XL in subchondral bone turnover and cartilage mineralization in humans via DIO2 and MGP functions. Previously, OPG-XL was shown to affect binding between RANKL and heparan sulphate (HS) resulting in loss of immobilized OPG-XL. Therefore, effects may be triggered by deficiency in the immobilization of OPG-XL Since the characteristic bidirectional pathophysiology of articular cartilage calcification accompanied by low subchondral bone mineralization is also a hallmark of OA pathophysiology, our results are likely extrapolated to common arthropathies.

Topics: Bone Remodeling; Calcinosis; Cartilage, Articular; Chondrocalcinosis; Humans; Induced Pluripotent Stem Cells; Mutation; Osteoprotegerin; RANK Ligand

The gene TNFRSF11B encodes for osteoprotegerin (OPG) and was recently identified as the CCAL1 locus associated with familial calcium pyrophosphate deposition disease (CPDD). While the CCAL1 OPG mutation (OPG-XL) was originally believed to be a gain-of-function mutation, loss of OPG activity causes arthritis-associated osteolysis in mice, which is likely related to excess subchondral osteoclast formation and/or activity. The purpose of the present study was to further explore the effect of OPG-XL in osteoclastogenesis.. The effects of recombinant OPG-XL and wild-type (WT) OPG were determined in monoculture and coculture models of RANKL-induced osteoclastogenesis. The effects of OPG-XL on osteoclast survival as well as on TRAIL-induced apoptosis were determined using standard in vitro assays and compared to WT OPG. The ability of OPG-XL and WT OPG to bind to osteoblasts was measured with enzyme-linked immunosorbent assay and flow cytometry using the osteoblastic MC3T3-E1 cell line.. OPG-XL was less effective than WT OPG at blocking RANKL-induced osteoclastogenesis in monoculture and coculture models. Osteoclast survival and inhibition of TRAIL-induced apoptosis were similar in the presence of OPG-XL and WT OPG. Compared to WT OPG, considerably less OPG-XL bound to cells.. These findings indicate that OPG-XL is a loss-of-function mutation as it relates to RANKL-mediated osteoclastogenesis, and thus may permit increased osteoclast numbers and heightened bone turnover. Further studies are necessary to demonstrate how this mutation contributes to arthritis in individuals carrying this mutation.

Topics: Animals; Bone Remodeling; Cell Line; Chondrocalcinosis; Coculture Techniques; Disease Models, Animal; Loss of Function Mutation; Mice; Osteogenesis; Osteoprotegerin

Mutations on chromosomes 5p (CCAL2) and 8q (CCAL1) have been linked to familial forms of calcium pyrophosphate deposition disease (CPDD). Mutations in the ANKH gene account for CCAL2, but the identity of CCAL1 has been elusive. Recently, a single Dutch kindred with a mutation in the Tumor Necrosis Factor Receptor Super Family member 11B (TNFRSF11B) gene coding for osteoprotegerin (OPG) was described as a gain-of-function mutation. Affected family members had premature generalized osteoarthritis (PGOA) and CPDD. As the TNFRSF11B gene is on 8q, we sought additional evidence that TNFRSF11B was CCAL1, and investigated potential disease mechanisms.. DNA from two novel PGOA/CPDD families was screened for sequence variants in the TNFRSF11B gene. Mutations were verified by genotype analysis of affected and unaffected family members. We also investigated effects of normal and mutant OPG on regulators of CPP crystal formation in porcine cartilage.. The identical TNFRSF11B mutation described in the Dutch family was present in two novel PGOA/CPDD families. ANKH was normal in affected patient fibroblasts. Exogenous OPG did not alter ANKH mRNA or protein levels, affect translocation of ANKH to the membrane, nor increase [pyrophosphate (PPi)] or other key regulators of CPDD.. We have firmly established the identity of CCAL1 as TNFRSF11B (OPG). Our findings suggest that this mutation produces disease in an ANKH-independent manner via novel mechanisms not primarily targeting cartilage. This work rationalizes further investigation of OPG pathway components as potential druggable targets for CPDD.

Topics: Adult; Chondrocalcinosis; Humans; Mutation; Osteoprotegerin; Pedigree

To identify pathogenic mutations that reveal underlying biological mechanisms driving osteoarthritis (OA).. Exome sequencing was applied to two distant family members with dominantly inherited early onset primary OA at multiple joint sites with chondrocalcinosis (familial generalised osteoarthritis, FOA). Confirmation of mutations occurred by genotyping and linkage analyses across the extended family. The functional effect of the mutation was investigated by means of a cell-based assay. To explore generalisability, mRNA expression analysis of the relevant genes in the discovered pathway was explored in preserved and osteoarthritic articular cartilage of independent patients undergoing joint replacement surgery.. We identified a heterozygous, probably damaging, read-through mutation (c.1205A=>T; p.Stop402Leu) in TNFRSF11B encoding osteoprotegerin that is likely causal to the OA phenotype in the extended family. In a bone resorption assay, the mutant form of osteoprotegerin showed enhanced capacity to inhibit osteoclastogenesis and bone resorption. Expression analyses in preserved and affected articular cartilage of independent OA patients showed that upregulation of TNFRSF11B is a general phenomenon in the pathophysiological process.. Albeit that the role of the molecular pathway of osteoprotegerin has been studied in OA, we are the first to demonstrate that enhanced osteoprotegerin function could be a directly underlying cause. We advocate that agents counteracting the function of osteoprotegerin could comply with new therapeutic interventions of OA.

Topics: Aged; Aged, 80 and over; Bone Resorption; Cell Differentiation; Chondrocalcinosis; Exome; Female; Genotype; Heterozygote; Humans; Male; Middle Aged; Mutation; Osteoarthritis; Osteoclasts; Osteoprotegerin; Pedigree; Phenotype

Bone destruction is a common feature of inflammatory arthritis and is mediated by osteoclasts, the only specialized cells to carry out bone resorption. Aberrant expression of receptor activator of nuclear factor kappa β ligand (RANKL), an inducer of osteoclast differentiation has been linked with bone pathology and the synovial fibroblast in rheumatoid arthritis (RA). In this manuscript, we challenge the current concept that an increase in RANKL expression governs osteoclastogenesis and bone destruction in autoimmune arthritis. We isolated human fibroblasts from RA, pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients and analyzed their RANKL/OPG expression profile and the capacity of their secreted factors to induce osteoclastogenesis. We determined a 10-fold increase of RANKL mRNA and protein in fibroblasts isolated from RA relative to PPA and OA patients. Peripheral blood mononuclear cells (PBMC) from healthy volunteers were cultured in the presence of RA, PPA and OA synovial fibroblast conditioned medium. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), F-actin ring formation and bone resorption assays. The formation of TRAP(+), VNR(+) multinucleated cells, capable of F-actin ring formation and lacunar resorption in synovial fibroblast conditioned medium cultures occured in the presence of osteoprotegerin (OPG) a RANKL antagonist. Osteoclasts did not form in these cultures in the absence of macrophage colony stimulating factor (M-CSF). Our data suggest that the conditioned medium of pure synovial fibroblast cultures contain inflammatory mediators that can induce osteoclast formation in human PBMC independently of RANKL. Moreover inhibition of the TNF or IL-6 pathway was not sufficient to abolish osteoclastogenic signals derived from arthritic synovial fibroblasts. Collectively, our data clearly show that alternate osteoclastogenic pathways exist in inflammatory arthritis and place the synovial fibroblast as a key regulatory cell in bone and joint destruction, which is a hallmark of autoimmune arthritis.

Topics: Acid Phosphatase; Actins; Arthritis, Rheumatoid; Bone Resorption; Cell Differentiation; Cells, Cultured; Chondrocalcinosis; Culture Media, Conditioned; Fibroblasts; Gene Expression Regulation; Humans; Integrin alphaVbeta3; Interleukin-6; Isoenzymes; Leukocytes, Mononuclear; Osteoarthritis; Osteoclasts; Osteoprotegerin; RANK Ligand; Signal Transduction; Synovial Fluid; Tartrate-Resistant Acid Phosphatase; Tumor Necrosis Factor-alpha