Probiotics Inhibit Cartilage Damage and Progression of Osteoarthritis in Mice.

Increasing interest has focussed on the possible role of alterations in the microbiome in the pathogenesis of metabolic disease, inflammatory disease, and osteoporosis. Here we examined the role of the microbiome in a preclinical model of osteoarthritis in mice subjected to destabilisation of medical meniscus (DMM). The intestinal microbiome was depleted by broad-spectrum antibiotics from 1 week before birth until the age of 6 weeks when mice were subjected reconstitution of the microbiome with faecal microbial transplant (FMT) followed by the administration of a mixture of probiotic strains Lacticaseibacillus paracasei 8700:2, Lactiplantibacillus plantarum HEAL9 and L. plantarum HEAL19 or vehicle. All mice were subjected to DMM at the age of 8 weeks. The severity of osteoarthritis was evaluated by histological analysis and effects on subchondral bone were investigated by microCT analyses. The combination of FMT and probiotics significantly inhibited cartilage damage at the medial femoral condyle such that the OARSI score was 4.64 ± 0.32 (mean ± sem) in the FMT and probiotic group compared with 6.48 ± 0.53 in the FMT and vehicle group (p = 0.007). MicroCT analysis of epiphyseal bone from the femoral condyle showed that the probiotic group had higher BV/TV, increased Tb.Th, and moderately thicker subchondral bone plates than the control group. There was no difference between groups in joint inflammation or in serum concentrations of inflammatory cytokines and chemokines. We conclude that treatment with probiotics following FMT in mice where the microbiome has been depleted inhibits DMM-induced cartilage damage and impacts on the structure of subchondral bone particularly at the femoral condyle. While further studies are required to elucidate the mechanism of action, our research suggests that these probiotics may represent a novel intervention for the treatment of osteoarthritis.

Association of cannabinoid receptor modulation with normal and abnormal skeletal remodelling: A systematic review and meta-analysis of in vitro, in vivo and human studies.

To address the inconsistent findings from studies that used different models to explore the role of classical cannabinoid type 1 (CB1) and 2 (CB2) receptors in skeletal remodelling, we searched Medline, Web of Science and Embase for relevant studies from inception to June 23, 2020. We identified 38 in vitro, 34 in vivo and 9 human studies. A meta-analysis of in vitro studies showed that exposure to the inverse-agonists AM251 (mean difference [MD]:-26.75, 95% confidence interval [CI]:-45.36,-8.14, p = 0.005), AM630 (standardised[std.] MD:-3.11, CI:-5.26,-0.97, p = 0.004; SR144528, std.MD:-4.88, CI -7.58,-2.18, p = 0.0004) and CBD (std.MD:-1.39, CI -2.64,-0.14, p = 0.03) is associated with reduced osteoclastogenesis, whereas the endocannabinoid 2-AG (std.MD:2.00, CI:0.11-3.89, p = 0.04) and CB2-selective agonist HU308 (MD:19.38, CI:11.75-27.01, p < 0.00001) were stimulatory. HU308 also enhanced osteoblast differentiation (std.MD:2.22, CI:0.95-3.50, p = 0.0006) and activity (std.MD:2.97, CI:1.22-4.71, p = 0.0008). In models of bone loss, CB1/2 deficiency enhanced peak bone volume (std.MD:3.70, CI:1.77-5.63, p = 0.0002) but reduced bone formation (std.MD:-0.54, CI:-0.90,-0.17, p = 0.004) in female mice. In male rats, CB1/2 deficiency (std.MD:2.31, CI:0.30-4.33, p = 0.02) and AM251 or CBD treatments (std.MD:2.19, CI:0.46-3.93, p = 0.01) enhanced bone volume. CB1/2 deficiency (std.MD:9.78, CI:4.96-14.61, p < 0.0001) and AM251 or AM630 treatments (std.MD:28.19, CI:19.13-37.25, p < 0.0001) were associated with osteoprotection. The CB2-selective agonists JWH133 and 4Q3C enhanced bone volume in arthritic rodents (std.MD:14.45, CI:2.08-26.81, p = 0.02). In human, CB2 SNPs (AA:rs2501431, MD:-0.28, CI:-0.55,-0.01, p = 0.04; CC:rs2501432, MD:-0.29, CI:-0.56,-0.02, p = 0.03) were associated with reduced bone mineral density, however the association of Marijuana use remains unclear. Thus, CB1/2 modulation is associated with altered bone metabolism, however findings are confounded by low study number and heterogenicity of models.

Targeted Inactivation of Rin3 Increases Trabecular Bone Mass by Reducing Bone Resorption and Favouring Bone Formation.

Common genetic variants at the RIN3 locus on chromosome 14q32 predispose to Paget's disease of bone (PDB) but the mechanisms by which they do so are unknown. Here, we analysed the skeletal phenotype of female mice with targeted inactivation of the mouse Rin3 gene (Rin3-/-) as compared with wild-type littermates. The Rin3-/- mice had higher trabecular bone volume (BV/TV%) compared with wild type. Mean ± standard deviation values at the distal femur at 8 weeks were 9.0 ± 2.5 vs. 7.0 ± 1.5 (p = 0.002) and at 52 weeks were 15.8 ± 9.5 vs. 8.5 ± 4.2 (p = 0.002). No differences were observed in femoral cortical bone parameters with the exception of marrow diameter which was significantly smaller in 52-week-old Rin3-/- mice compared to wild type: (0.43 mm ± 0.1 vs. 0.57 mm ± 0.2 (p = 0.001). Bone histomorphometry showed a lower osteoclast surface / bone surface (Oc.S/BS%) at 8 weeks in Rin3-/- mice compared to wild type (24.1 ± 4.7 vs. 29.7 ± 6.6; p = 0.025) but there were no significant differences in markers of bone formation at this time. At 52 weeks, Oc.S/BS did not differ between genotypes but single labelled perimeter (SL.Pm/B.Pm (%)) was significantly higher in Rin3-/- mice (24.4 ± 6.4 vs. 16.5 ± 3.8, p = 0.003). We conclude that Rin3 negatively regulates trabecular bone mass in mice by inhibiting osteoclastic bone resorption and favouring bone formation. Our observations also suggest that the variants that predispose to PDB in humans probably do so by causing a gain-in-function of RIN3.

JZL184, A Monoacylglycerol Lipase Inhibitor, Induces Bone Loss in a Multiple Myeloma Model of Immunocompetent Mice.

Multiple myeloma (MM) patients develop osteolysis characterised by excessive osteoclastic bone destruction and lack of osteoblast bone formation. Pharmacological manipulation of monoacylglycerol lipase (MAGL), an enzyme responsible for the degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced skeletal tumour burden and osteolysis associated with osteosarcoma and advanced breast and prostate cancers. MM and hematopoietic, immune and bone marrow cells express high levels of type 2 cannabinoid receptor and osteoblasts secrete 2-AG. However, the effects of MAGL manipulation on MM have not been investigated. Here, we report that treatment of pre-osteoclasts with non-cytotoxic concentrations of JZL184, a verified MAGL inhibitor, enhanced MM- and RANKL-induced osteoclast formation and size in vitro. Exposure of osteoblasts to JZL184 in the presence of MM cell-derived factors reduced osteoblast growth but had no effect on the ability of these cells to mature or form bone nodules. In vivo, administration of JZL184 induced a modest, yet significant, bone loss at both trabecular and cortical compartments of long bones of immunocompetent mice inoculated with the syngeneic 5TGM1-GFP MM cells. Notably, JZL184 failed to inhibit the in vitro growth of a panel of mouse and human MM cell lines, or reduce tumour burden in mice. Thus, MAGL inhibitors such as JZL184 can exacerbate MM-induced bone loss.

Liver-derived IGF-I is not required for protection against osteoarthritis in male mice.

Insulin-like growth factor-I (IGF-I) is anabolic for cartilage and important for cartilage integrity, which might suggest a connection between IGF-I and osteoarthritis (OA) development. However, the results of studies performed so far are conflicting, and we aimed to clarify the role of endocrine IGF-I in rodent OA. Male mice with inducible inactivation of circulating, liver-derived IGF-I (LI-IGF-I-/- mice, serum IGF-I reduced by ~80%) were used. Experimental OA was induced in young adult LI-IGF-I-/- and control mice by destabilization of the medial meniscus (DMM); age-related OA was also evaluated in 1-yr-old mice. DMM-operated LI-IGF-I-/- mice had thinner lateral subchondral bone plate in tibia compared with control mice, whereas osteophyte volume and articular cartilage damage were unaffected at the medial side of the DMM knee. However, the control mice but not the LI-IGF-I-/- mice also developed mild OA on the lateral side of the DMM knee compared with the unoperated knee. One-year-old LI-IGF-I-/- mice had lower mid-diaphyseal cortical bone area than the 1-yr-old control mice, whereas analyses of joint tissues displayed smaller osteophyte volume and thicker calcified cartilage than the control mice. There was no difference in OA severity in the articular cartilage between old LI-IGF-I-/- and control mice. Our study is the first to investigate whether there is an association between circulating IGF-I and OA in mice. We conclude that, although there is an ~80% reduction of circulating IGF-I and a decrease in cortical bone in male LI-IGF-I-/- mice, cartilage damage is clearly not intensified and may instead be slightly reduced.

Raman spectroscopy as a predictive tool for monitoring osteoporosis therapy in a rat model of postmenopausal osteoporosis.

Pharmacological therapy of osteoporosis reduces bone loss and risk of fracture in patients. Modulation of bone mineral density cannot explain all effects. Other aspects of bone quality affecting fragility and ways to monitor them need to be better understood. Keratinous tissue acts as surrogate marker for bone protein deterioration caused by oestrogen deficiency in rats. Ovariectomised rats were treated with alendronate (ALN), parathyroid hormone (PTH) or estrogen (E2). MicroCT assessed macro structural changes. Raman spectroscopy assessed biochemical changes. Micro CT confirmed that all treatments prevented ovariectomy-induced macro structural bone loss in rats. PTH induced macro structural changes unrelated to ovariectomy. Raman analysis revealed ALN and PTH partially protect against molecular level changes to bone collagen (80% protection) and mineral (50% protection) phases. E2 failed to prevent biochemical change. The treatments induced alterations unassociated with the ovariectomy; increased beta sheet with E2, globular alpha helices with PTH and fibrous alpha helices with both ALN and PTH. ALN is closest to maintaining physiological status of the animals, while PTH (comparable protective effect) induces side effects. E2 is unable to prevent molecular level changes associated with ovariectomy. Raman spectroscopy can act as predictive tool for monitoring pharmacological therapy of osteoporosis in rodents. Keratinous tissue is a useful surrogate marker for the protein related impact of these therapies.The results demonstrate utility of surrogates where a clear systemic causation connects the surrogate to the target tissue. It demonstrates the need to assess broader biomolecular impact of interventions to examine side effects.

Raman spectroscopy predicts the link between claw keratin and bone collagen structure in a rodent model of oestrogen deficiency.

Osteoporosis is a common disease characterised by reduced bone mass and an increased risk of fragility fractures. Low bone mineral density is known to significantly increase the risk of osteoporotic fractures, however, the majority of non-traumatic fractures occur in individuals with a bone mineral density too high to be classified as osteoporotic. Therefore, there is an urgent need to investigate aspects of bone health, other than bone mass, that can predict the risk of fracture. Here, we successfully predicted association between bone collagen and nail keratin in relation to bone loss due to oestrogen deficiency using Raman spectroscopy. Raman signal signature successfully discriminated between ovariectomised rats and their sham controls with a high degree of accuracy for the bone (sensitivity 89%, specificity 91%) and claw tissue (sensitivity 89%, specificity 82%). When tested in an independent set of claw samples the classifier gave 92% sensitivity and 85% specificity. Comparison of the spectral changes occurring in the bone tissue with the changes occurring in the keratin showed a number of common features that could be attributed to common changes in the structure of bone collagen and claw keratin. This study established that systemic oestrogen deficiency mediates parallel structural changes in both the claw (primarily keratin) and bone proteins (primarily collagen). This strengthens the hypothesis that nail keratin can act as a surrogate marker of bone protein status where systemic processes induce changes.

Targeted sequencing of the Paget's disease associated 14q32 locus identifies several missense coding variants in RIN3 that predispose to Paget's disease of bone.

Paget's disease of bone (PDB) is a common disorder with a strong genetic component characterized by increased but disorganized bone remodelling. Previous genome-wide association studies identified a locus on chromosome 14q32 tagged by rs10498635 which was significantly associated with susceptibility to PDB in several European populations. Here we conducted fine-mapping and targeted sequencing of the candidate locus to identify possible functional variants. Imputation in 741 PDB patients and 2699 controls confirmed that the association was confined to a 60 kb region in the RIN3 gene and conditional analysis adjusting for rs10498635 identified no new independent signals. Sequencing of the RIN3 gene identified a common missense variant (p.R279C) that was strongly associated with the disease (OR = 0.64; P = 1.4 × 10(-9)), and was in strong linkage disequilibrium with rs10498635. A further 13 rare missense variants were identified, seven of which were novel and detected only in PDB cases. When combined, these rare variants were over-represented in cases compared with controls (OR = 3.72; P = 8.9 × 10(-10)). Most rare variants were located in a region that encodes a proline-rich, intrinsically disordered domain of the protein and many were predicted to be pathogenic. RIN3 was expressed in bone tissue and its expression level was ∼10-fold higher in osteoclasts compared with osteoblasts. We conclude that susceptibility to PDB at the 14q32 locus is mediated by a combination of common and rare coding variants in RIN3 and suggest that RIN3 may contribute to PDB susceptibility by affecting osteoclast function.

Bone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast Cancer-induced Osteolysis.

The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.

Genetic background modifies the effects of type 2 cannabinoid receptor deficiency on bone mass and bone turnover.

Cannabinoid receptors and their ligands play significant roles in regulating bone metabolism. Previous studies of type 1 cannabinoid receptor-deficient mice have shown that genetic background influences the skeletal phenotype. Here, we investigated the effects of genetic background on the skeletal phenotype of mice with type 2 cannabinoid receptor deficiency (Cnr2 (-/-)). We studied Cnr2 (-/-) mice on a CD1 background and compared the findings with those previously reported in Cnr2 (-/-) C57BL/6 mice. Young female Cnr2 (-/-) CD1 mice had low bone turnover and high trabecular bone mass compared with wild-type (WT), contrasting with the situation in Cnr2 (-/-) C57BL/6 mice where trabecular bone mass has been reported to be similar to WT. The Cnr2 (-/-) CD1 mice lost more trabecular bone at the tibia with age than WT due to reduced bone formation, and at 12 months there was no difference in trabecular bone volume between genotypes. This differs from the phenotype previously reported in C57BL/6 Cnr2 (-/-) mice, where bone turnover is increased and bone mass reduced with age. There were no substantial differences in skeletal phenotype between Cnr2 (-/-) and WT in male mice. Cortical bone phenotype was similar in Cnr2 (-/-) and WT mice of both genders. Deficiency of Cnr2 has site- and gender-specific effects on the skeleton, mainly affecting trabecular bone, which are influenced by genetic differences between mouse strains. Further evaluation of the pathways responsible might yield new insights into the mechanisms by which cannabinoid receptors regulate bone metabolism.

Modulation of strain-specific differences in gene expression by cannabinoid type 2 receptor deficiency.

Previous studies have shown that the skeletal consequences of cannabinoid receptor deficiency differ in different strains of mice. In order to explore the mechanisms responsible, we analysed global gene expression in bone from wild-type CD1 mice and littermates with targeted inactivation of the type 2 cannabinoid receptor (Cnr2 (-/-)) and compared the results with those obtained from a similar analysis of wild-type and Cnr2 (-/-) C57BL/6 mice. Trabecular bone volume was increased in Cnr2 (-/-) CD1 mice compared with wild-type littermates but decreased in Cnr2 (-/-) C57BL/6 mice. Microarray analysis identified 354 genes in which substantial differences in gene expression (>1.5-fold) were observed that were specifically affected by Cnr2 deficiency. Bioinformatic analysis of data from wild-type mice of each strain revealed Cnr2-dependent differences in expression of genes clustering within the gene ontology (GO) terms immune response (p < 0.0001), positive regulation of response to stimulus (p < 0.0001), nucleotide binding (p = 0.002), and ribonucleotide binding (p = 0.003). Bioinformatic analysis of data from Cnr2 (-/-) mice of each strain revealed associations between GO terms corresponding to the extracellular region (p = 0.002), the cell surface (p = 0.02), antigen binding (p = 0.03), external side of plasma membrane (p = 0.04), and regulation of the force of heart contraction (p = 0.04). We conclude that Cnr2 deficiency affects expression of a large number of genes in different strains of mice, and that these differences are likely to be responsible in part for the differences in skeletal phenotype that we and others have observed in mice with defective cannabinoid receptor signalling in different genetic backgrounds.

The Effect of Probiotics on the Management of Pain and Inflammation in Osteoarthritis: A Systematic Review and Meta-Analysis of Clinical Studies.

Osteoarthritis (OA) is one of the most common musculoskeletal disorders. Recently, research has focused on the role of intestinal microbiome dysbiosis in OA. The aim of this study was to systematically review randomized intervention clinical studies investigating the effect of probiotics on the management of OA-related pain and inflammation. Pre-clinical studies and non-randomized trials were excluded. A literature search was conducted using MEDLINE, EMBASE, and Web of Science. Study quality was assessed with the Cochrane risk of bias (RoB2) tool and the Risk of Bias in N-of-1 Trials (RoBiNT) scale. RevMan was used for the meta-analysis. Outcome measures assessed self-reported pain, stiffness and impediment, and serum hs-CRP. Three studies, with 501 participants, were considered eligible for qualitative synthesis and meta-analysis. A significant reduction in symptoms across all outcomes measured, except stiffness, was evident with Lactobacillus casei Shirota. However, all other probiotics reviewed did not seem to have any effect on the measured outcomes. Pre-clinical evidence, along with the RCTs reviewed, suggests that probiotics of the Lactobacillus strains might be of use for managing pain and inflammation in OA. Considering the small number of studies included in the present review and the possible risk of bias, we conclude that further studies on the role of probiotics in humans with OA are warranted.

Classical cannabinoid receptors as target in cancer-induced bone pain: a systematic review, meta-analysis and bioinformatics validation.

To test the hypothesis that genetic and pharmacological modulation of the classical cannabinoid type 1 (CB1) and 2 (CB2) receptors attenuate cancer-induced bone pain, we searched Medline, Web of Science and Scopus for relevant skeletal and non-skeletal cancer studies from inception to July 28, 2022. We identified 29 animal and 35 human studies. In mice, a meta-analysis of pooled studies showed that treatment of osteolysis-bearing males with the endocannabinoids AEA and 2-AG (mean difference [MD] - 24.83, 95% confidence interval [95%CI] - 34.89, - 14.76, p < 0.00001) or the synthetic cannabinoid (CB) agonists ACPA, WIN55,212-2, CP55,940 (CB1/2-non-selective) and AM1241 (CB2-selective) (MD - 28.73, 95%CI - 45.43, - 12.02, p = 0.0008) are associated with significant reduction in paw withdrawal frequency. Consistently, the synthetic agonists AM1241 and JWH015 (CB2-selective) increased paw withdrawal threshold (MD 0.89, 95%CI 0.79, 0.99, p < 0.00001), and ACEA (CB1-selective), AM1241 and JWH015 (CB2-selective) reduced spontaneous flinches (MD - 4.85, 95%CI - 6.74, - 2.96, p < 0. 00001) in osteolysis-bearing male mice. In rats, significant increase in paw withdrawal threshold is associated with the administration of ACEA and WIN55,212-2 (CB1/2-non-selective), JWH015 and AM1241 (CB2-selective) in osteolysis-bearing females (MD 8.18, 95%CI 6.14, 10.21, p < 0.00001), and treatment with AM1241 (CB2-selective) increased paw withdrawal thermal latency in males (mean difference [MD]: 3.94, 95%CI 2.13, 5.75, p < 0.0001), confirming the analgesic capabilities of CB1/2 ligands in rodents. In human, treatment of cancer patients with medical cannabis (standardized MD - 0.19, 95%CI - 0.35, - 0.02, p = 0.03) and the plant-derived delta-9-THC (20 mg) (MD 3.29, CI 2.24, 4.33, p < 0.00001) or its synthetic derivative NIB (4 mg) (MD 2.55, 95%CI 1.58, 3.51, p < 0.00001) are associated with reduction in pain intensity. Bioinformatics validation of KEGG, GO and MPO pathway, function and process enrichment analysis of mouse, rat and human data revealed that CB1 and CB2 receptors are enriched in a cocktail of nociceptive and sensory perception, inflammatory, immune-modulatory, and cancer pathways. Thus, we cautiously conclude that pharmacological modulators of CB1/2 receptors show promise in the treatment of cancer-induced bone pain, however further assessment of their effects on bone pain in genetically engineered animal models and cancer patients is warranted.

The Role of Nutrition in Osteoarthritis Development.

Osteoarthritis (OA) prevalence has increased 113% since 1990, and currently more than half a billion people worldwide are living with this slowly progressing, degenerative joint disease [...].

Effects of zoledronic acid on osteosarcoma progression and metastasis: systematic review and meta-analysis.

Zoledronic Acid (ZA) has been shown to inhibit Osteosarcoma (OSA) progression in preclinical studies. However, the use of ZA as an intervention for OSA treatment and management remains controversial. A systematic review and meta-analysis of randomized-controlled trials comparing the use of ZA with standard treatment vs. standard treatment alone for OSA patients after resection was conducted. Primary outcomes assessed event-free survival (EFS) and overall survival (OS) rates, while secondary outcomes assessed impact of ZA on metastatic spread, histological response and adverse events occurrence. A literature search was conducted using EMBASE, MEDLINE, and the Cochrane Central Register of Controlled Trials. The Cochrane risk of bias tool (version 2) was used to assess trial quality. RevMan v5.4 was used for the meta-analysis. The between-trial heterogeneity was assessed using the Chi2 test and the I2 statistic and the GRADE methodology was utilized to assess certainty of evidence. Two studies were considered eligible for qualitative synthesis and meta-analysis. ZA had no benefit on EFS (HR, 0.95; 95% CI, 0.48-1.88; p-value 0.88), however, when compared to standard treatment it reduced OS (HR, 1.98; 95% CI, 1.49-2.64; p-value < 0.00001). ZA did not deter lung metastasis (RR, 2.56; 95% CI, 0.35-18.60; p-value 0.35), and neither did it increase good histological response (RR, 0.97; 95% CI, 0.90-1.05; p-value 0.48). ZA treatment was associated with higher risk of adverse events. Based on existing data, the use of ZA as adjuvant therapy is not recommended for the treatment of OSA patients.

TRAF6 as a potential target in advanced breast cancer: a systematic review, meta-analysis, and bioinformatics validation.

TRAF6 has emerged as a key regulator of breast cancer (BCa). However, the TRAF family constitutes of seven members that exhibit distinct and overlapping functions. To explore which TRAF represents a potential druggable target for BCa treatment, we searched Medline, Web of Science and Scopus for relevant studies from inception to June 27, 2021. We identified 14 in vitro, 11 in vivo and 4 human articles. A meta-analysis of pharmacological studies showed that in vitro inhibition of TRAF2/4 (mean difference (MD): - 57.49, 95% CI: - 66.95, - 48.02, P < 0.00001) or TRAF6 (standard(Std.)MD: - 4.01, 95% CI: - 5.75, - 2.27, P < 0.00001) is associated with reduction in BCa cell migration. Consistently, inhibition of TRAF2/4 (MD: - 51.08, 95% CI: - 64.23, - 37.94, P < 0.00001) and TRAF6 (Std.MD: - 2.80, 95% CI: - 4.26, - 1.34, P = 0.0002) is associated with reduced BCa cell invasion, whereas TRAF2/4 inhibition (MD: - 40.54, 95% CI: - 52.83, - 28.26, P < 0.00001) is associated with reduced BCa cell adhesion. Interestingly, only inhibition of TRAF6 (MD: - 21.46, 95% CI: - 30.40, - 12.51, P < 0.00001) is associated with reduced cell growth. In animal models of BCa, administration of pharmacological inhibitors of TRAF2/4 (Std.MD: - 3.36, 95% CI: - 4.53, - 2.18, P < 0.00001) or TRAF6 (Std.MD: - 4.15, 95% CI: - 6.06, - 2.24, P < 0.0001) in mice is associated with reduction in tumour burden. In contrast, TRAF6 inhibitors (MD: - 2.42, 95% CI: - 3.70, - 1.14, P = 0.0002) reduced BCa metastasis. In BCa patients, high expression of TRAF6 (Hazard Ratio: 1.01, CI: 1.01, 1.01, P < 0.00001) is associated with poor survival rate. Bioinformatics validation of clinical and pathway and process enrichment analysis in BCa patients confirmed that gain/amplification of TRAF6 is associated with secondary BCa in bone (P = 0.0079), and poor survival rate (P < 0.05). Overall, TRAF6 inhibitors show promise in the treatment of metastatic BCa. However, low study number and scarcity of evidence from animal and human studies may limit the translation of present findings into clinical practice.

Anti-inflammatory, but not osteoprotective, effect of the TRAF6/CD40 inhibitor 6877002 in rodent models of local and systemic osteolysis.

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1ß-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates.

Dexamethasone Administration in Mice Leads to Less Body Weight Gain over Time, Lower Serum Glucose, and Higher Insulin Levels Independently of NRF2.

Glucocorticoids are used widely on a long-term basis in autoimmune and inflammatory diseases. Their adverse effects include the development of hyperglycemia and osteoporosis, whose molecular mechanisms have been only partially studied in preclinical models. Both these glucocorticoid-induced pathologies have been shown to be mediated at least in part by oxidative stress. The transcription factor nuclear erythroid factor 2-like 2 (NRF2) is a central regulator of antioxidant and cytoprotective responses. Thus, we hypothesized that NRF2 may play a role in glucocorticoid-induced metabolic disease and osteoporosis. To this end, WT and Nrf2 knockout (Nrf2KO) mice of both genders were treated with 2 mg/kg dexamethasone or vehicle 3 times per week for 13 weeks. Dexamethasone treatment led to less weight gain during the treatment period without affecting food consumption, as well as to lower glucose levels and high insulin levels compared to vehicle-treated mice. Dexamethasone also reduced cortical bone volume and density. All these effects of dexamethasone were similar between male and female mice, as well as between WT and Nrf2KO mice. Hepatic NRF2 signaling and gluconeogenic gene expression were not affected by dexamethasone. A 2-day dexamethasone treatment was also sufficient to increase insulin levels without affecting body weight and glucose levels. Hence, dexamethasone induces hyperinsulinemia, which potentially leads to decreased glucose levels, as well as osteoporosis, both independently of NRF2.

Conditional deletion of E11/Podoplanin in bone protects against ovariectomy-induced increases in osteoclast formation and activity.

E11/Podoplanin (Pdpn) is implicated in early osteocytogenesis and the formation of osteocyte dendrites. This dendritic network is critical for bone modelling/remodelling, through the production of receptor activator of nuclear factor κ B (RANK)-ligand (RANKL). Despite this, the role of Pdpn in the control of bone remodelling is yet to be established in vivo. Here we utilised bone-specific Pdpn conditional knockout mice (cKO) to examine the role of Pdpn in the bone loss associated with ovariectomy (OVX). MicroCT revealed that Pdpn deletion had no significant effect on OVX-induced changes in trabecular microarchitecture. Significant differences between genotypes were observed in the trabecular pattern factor (P<0.01) and structure model index (P<0.01). Phalloidin staining of F-actin revealed OVX to induce alterations in osteocyte morphology in both wild-type (WT) and cKO mice. Histological analysis revealed an expected significant increase in osteoclast number in WT mice (P<0.01, compared with sham). However, cKO mice were protected against such increases in osteoclast number. Consistent with this, serum levels of the bone resorption marker Ctx were significantly increased in WT mice following OVX (P<0.05), but were unmodified by OVX in cKO mice. Gene expression of the bone remodelling markers Rank, Rankl, Opg and Sost were unaffected by Pdpn deletion. Together, our data suggest that an intact osteocyte dendritic network is required for sustaining osteoclast formation and activity in the oestrogen-depleted state, through mechanisms potentially independent of RANKL expression. This work will enable a greater understanding of the role of osteocytes in bone loss induced by oestrogen deprivation.