TNF-induced osteoclastogenesis and inflammatory bone resorption are inhibited by transcription factor RBP-J

Authors

Baohong Zhao, Shannon N. Grimes, Susan Li, Xiaoyu Hu, and Lionel B. Ivashkiv

Abstract

Tumor necrosis factor (TNF) plays a key role in the pathogenesis of inflammatory bone resorption and associated morbidity in diseases such as rheumatoid arthritis and periodontitis. Mechanisms that regulate the direct osteoclastogenic properties of TNF to limit pathological bone resorption in inflammatory settings are mostly unknown. Here, we show that the transcription factor recombinant recognition sequence binding protein at the Jκ site (RBP-J) strongly suppresses TNF-induced osteoclastogenesis and inflammatory bone resorption, but has minimal effects on physiological bone remodeling. Myeloid-specific deletion of RBP-J converted TNF into a potent osteoclastogenic factor that could function independently of receptor activator of NF-κB (RANK) signaling. In the absence of RBP-J, TNF effectively induced osteoclastogenesis and bone resorption in RANK-deficient mice. Activation of RBP-J selectively in osteoclast precursors suppressed inflammatory osteoclastogenesis and arthritic bone resorption. Mechanistically, RBP-J suppressed induction of the master regulator of osteoclastogenesis (nuclear factor of activated T cells, cytoplasmic 1) by attenuating c-Fos activation and suppressing induction of B lymphocyte–induced maturation protein-1, thereby preventing the down-regulation of transcriptional repressors such as IRF-8 that block osteoclast differentiation. Thus, RBP-J regulates the balance between activating and repressive signals that regulate osteoclastogenesis. These findings identify RBP-J as a key upstream negative regulator of osteoclastogenesis that restrains excessive bone resorption in inflammatory settings.

Link to Article

http://dx.doi.org/10.1084/jem.20111566

A murine model of neurofibromatosis type 1 tibial pseudarthrosis featuring proliferative fibrous tissue and osteoclast-like cells

Authors

Jad El-Hoss MSc1,2,*, Kate Sullivan PhD1,2, Tegan Cheng1, Nicole YC Yu BEng(Hons)1, Justin D Bobyn BSc, MBBS2, Lauren Peacock1, Kathy Mikulec1, Paul Baldock PhD3, Ian E Alexander MBBS, FRACP, PhD2, Aaron Schindeler PhD1,2, David G Little MBBS, FRACS(Orth), PhD

Abstract

Neurofibromatosis type 1 is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue specific lesions associated with local double-inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre-expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1flox/flox and Nf1flox/- mice. The effects of the presence of Nf1null cells were extensively examined. Cultured Nf1null committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with rhBMP-2 treatment. Similarly, Nf1null inducible osteoprogenitors obtained from Nf1 mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1flox/flox and Nf1flox/- mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1flox/flox and Nf1flox/- mice. Histological analyses showed invasion of the Nf1null fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upwards of 10-fold in Nf1null fractures and BrdU staining in closed fractures showed increased numbers of proliferating cells. In Nf1null fractures, TRAP+ cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds.

Link to Article

htt://dx.doi.org/10.1002/jbmr.528/

Mice lacking Nf1 in osteochondroprogenitor cells display skeletal dysplasia similar to patients with neurofibromatosis type I

Authors

Weixi Wang, Jeffrey S. Nyman, Koichiro Ono, David A. Stevenson, Xiangli Yang and Florent Elefteriou

Abstract

Mutations in NF1 cause neurofibromatosis type I (NF1), a disorder characterized, among other clinical manifestations, by generalized and focal bony lesions. Dystrophic scoliosis and tibial pseudoarthrosis are the most severe skeletal manifestations for which treatment is not satisfactory, emphasizing the dearth of knowledge related to the biology of NF1 in bone cells. Using reporter mice, we report here that the mouse Col2α1-Cre promoter is active in chondrocytes but also in adult bone marrow osteoprogenitors giving rise to osteoblasts. Based on this finding, we crossed the Col2α1-Cre transgenic and Nf1flox/flox mice to determine whether loss of Nf1 in axial and appendicular osteochondroprogenitors recapitulates the skeletal abnormalities of NF1 patients. By microtomographic and X-rays studies, we show that Nf1Col2-/- mice display progressive scoliosis and kyphosis, tibial bowing, and abnormalities in skull and anterior chest wall formation. These defects were accompanied by a low bone mass phenotype, high bone cortical porosity, osteoidosis, increased osteoclastogenesis, and decreased osteoblast number, as quantified by histomorphometry and 3D-microtomography. Loss of Nf1 in osteochondroprogenitors also caused severe short stature and intervertebral disc defects. Blockade of the RAS/ERK activation characteristic of Nf1-/- osteoprogenitors by lovastatin during embryonic development could attenuate the increased cortical porosity observed in mutant pups. These data and the skeletal similarities between this mouse model and NF1 patients thus suggest that activation of the RAS/ERK pathway by Nf1 loss-of-function in osteochondroprogenitors is responsible for the vertebral and tibia lesions in NF1 patients, and that this molecular signature may represent a good therapeutic target.

Link to Article

http://dx.doi.org/10.1093/hmg/ddr310

Strontium ranelate inhibits key factors affecting bone remodeling in human osteoarthritic subchondral bone osteoblasts

Authors

Steeve Kwan Tata, Jean-Pierre Pelletiera, François Mineaua, Judith Carona, and Johanne Martel-Pelletier

Abstract

In osteoarthritis (OA) the progression of cartilage degeneration has been associated with remodeling of the subchondral bone. Human OA subchondral bone osteoblasts were shown to have an abnormal phenotype and altered metabolism leading to an abnormal resorptive process. Bone resorption is suggested to occur, at least in part, through the increased levels of two proteolytic enzymes, MMP-2 and MMP-9, and RANKL, which are mainly produced by osteoblasts. In this study, we investigated in human OA subchondral bone osteoblasts the modulatory effect of strontium ranelate on the above key factors. Human subchondral bone osteoblasts were cultured in a medium containing 0.1, 1 and 2 mM of strontium ranelate for 18 h for mRNA and 72 h for protein determination. The effect of strontium ranelate was evaluated on the expression (qPCR) of MMP-2, MMP-9, OPG, RANKL (total), RANKL-1, and RANKL-3, on the production of OPG (ELISA), membranous RANKL (flow cytometry), and MT1-MMP, ADAM17, and ADAM19 (Western blot). After incubation of osteoblasts with pre-osteoclasts (i.e., differentiated human peripheral blood mononuclear cells), the resorbed surface was measured using a sub-micron synthetic calcium phosphate thin film. Firstly, the expression levels of MMP-2, MMP-9, OPG, and RANKL were determined in normal and OA subchondral bone osteoblasts. As expected, the gene expression of MMP-9 and RANKL were not detectable in normal cells, whereas MMP-2 was very low but detectable and OPG demonstrated high gene expression. Further experiments looking at the effect of strontium ranelate on expression levels, except for OPG, were performed only on the OA subchondral bone osteoblasts. In OA cells, the expression levels of MMP-2 and MMP-9 were significantly decreased by strontium ranelate at 1 mM (p ≤ 0.005, p ≤ 0.02, respectively) and 2 mM (p ≤ 0.003, p ≤ 0.007), and for MMP-9 only at 0.1 mM (p ≤ 0.05). In normal cells, the expression of OPG was increased with strontium ranelate at 2 mM, and in OA both the expression (p ≤ 0.02) and synthesis (p ≤ 0.002) of OPG were significantly increased with strontium ranelate at 1 and 2 mM. RANKL (total) as well as the isoforms RANKL-1 and RANKL-3 were significantly increased by strontium ranelate at 1 and 2 mM. Of note, it is known that the different RANKL isoforms differentially regulate RANKL membranous localization: RANKL-3, in contrast to RANKL-1, prevents such membranous localization. This is reflected by the significant (p ≤ 0.02) reduction in the level of membranous RANKL by strontium ranelate at 2 mM. This latter finding was not likely to be related to a proteolytic cleavage of membranous RANKL, as the enzymes known to cleave it, MT1-MMP, ADAM17 and ADAM19, were unaffected by strontium ranelate. In addition, OA osteoblasts treated with strontium ranelate induced a significant (p ≤ 0.002) decrease in resorbed surface at the three tested concentrations. This study provides new insights into the mode of action of strontium ranelate on the metabolism of human OA subchondral bone osteoblasts. These data suggest that strontium ranelate may exert a positive effect on OA pathophysiology by inhibiting, in these cells, the synthesis of key factors leading to bone resorption, a feature associated with the OA process.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.06.005

IL-23 Is Critical for Induction of Arthritis, Osteoclast Formation, and Maintenance of Bone Mass

Authors

Iannis E. Adamopoulos, Marlowe Tessmer, Cheng-Chi Chao, Sarvesh Adda, Dan Gorman, Mary Petro, Chuan-Chu Chou, Robert H. Pierce, Wei Yao, Nancy E. Lane, Drake Laface, and Edward P. Bowman

Abstract

The role of IL-23 in the development of arthritis and bone metabolism was studied using systemic IL-23 exposure in adult mice via hydrodynamic delivery of IL-23 minicircle DNA in vivo and in mice genetically deficient in IL-23. Systemic IL-23 exposure induced chronic arthritis, severe bone loss, and myelopoiesis in the bone marrow and spleen, which resulted in increased osteoclast differentiation and systemic bone loss. The effect of IL-23 was partly dependent on CD4+ T cells, IL-17A, and TNF, but could not be reproduced by overexpression of IL-17A in vivo. A key role in the IL-23–induced arthritis was made by the expansion and activity of myeloid cells. Bone marrow macrophages derived from IL-23p19−/− mice showed a slower maturation into osteoclasts with reduced tartrate-resistant acid phosphatase-positive cells and dentine resorption capacity in in vitro osteoclastogenesis assays. This correlated with fewer multinucleated osteoclast-like cells and more trabecular bone volume and number in 26-wk-old male IL-23p19−/− mice compared with control animals. Collectively, our data suggest that systemic IL-23 exposure induces the expansion of a myeloid lineage osteoclast precursor, and targeting IL-23 pathway may combat inflammation-driven bone destruction as observed in rheumatoid arthritis and other autoimmune arthritides.

Link to Article

http://dx.doi.org/10.4049/jimmunol.1190033

Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model

Authors

Maxim Moreau, Pascale Rialland, Jean-Pierre Pelletier, Johanne Martel-Pelletier, Daniel Lajeunesse, Christelle Boileau, Judith Caron, Diane Frank, Bertrand Lussier, Jerome RE del Castillo, Guy Beauchamp, Dominique Gauvin, Thierry Bertaim, Dominique Thibaud and Eric Troncy

Abstract

The aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA). Baseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving 4 TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for 8 weeks. At baseline, week 4 and week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (MMP-1, MMP-13, and ADAMTS5) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE2 and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an alpha-threshold of 0.05. A better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE2 (P = 0.02), nitrites/ nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone. Tiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.

Link to Article

http://dx.doi.org/10.1186/ar3373