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Inhibition of early response genes prevents changes in global joint metabolomic profiles in mouse post-traumatic osteoarthritis.

ABSTRACT: OBJECTIVE:Although joint injury itself damages joint tissues, a substantial amount of secondary damage is mediated by the cellular responses to the injury. Cellular responses include the production and activation of proteases (MMPs, ADAMTSs, Cathepsins), and the production of inflammatory cytokines. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Our objective was to determine whether inhibition of cdk9-dependent early response gene activation affects changes in the joint metabolome. DESIGN:To model post-traumatic osteoarthritis, we subjected mice to non-invasive Anterior Cruciate Ligament (ACL)-rupture joint injury. Following injury, mice were treated with flavopiridol - a potent and selective inhibitor of Cdk9 kinase activity - to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1 h after injury. RESULTS:We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism, anandamide, and others. Inhibition of primary response gene activation immediately after injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites. CONCLUSIONS:Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses. We identified two sets of metabolites that change acutely after joint injury: those that require transcription of primary response genes, and those that do not. These data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury, which may offer novel targets for cell-mediated secondary joint damage.

SUBMITTER: Haudenschild DR

PROVIDER: S-EPMC6391201 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Project description:The prevalence of osteoarthritis (OA) varies between joints. Cartilage in eight different joints was evaluated to elucidate the disparate susceptibilities between joints to post-traumatic OA (PTOA) and provide evidence for joint-specific clinical treatments. The hypothesis was that cartilage in different joints would have varying cell death and anabolic gene expression profiles after injury.Adult equine cartilage explants were harvested from shoulder (SH), elbow (EL), carpal (CA), metacarpophalangeal (MC), patellofemoral (FP), tarsal (TA), metatarsophalangeal (MT), and proximal interphalangeal (PP) joints, and injured by loading with 30 MPa within 1 s. Fractional dissipated energy, cell density, cell death, and gene expression were quantified.PP had the highest fractional dissipated energy (94%, 95% confidence interval [CI] 88 to 101%). Cell density was highest in the superficial zone in all samples, with MC and MT having the highest peak density. Injured samples had significantly increased cell death (13.5%, 95% CI 9.1 to 17.9%) than non-injured samples (6.8%, 95% CI 2.5 to 11.1%, P = 0.016); however, cell death after injury was not significantly different between joints. Gene expression was significantly different between joints. CD-RAP expression in normal cartilage was lowest in FP (Cp = 21, 95% CI -80 to 122). After injury, the change in CD-RAP expression increased and was highest in FP (147% relative increase after injury, 95% CI 64 to 213).Different joints have different baseline characteristics, including cell density and gene expression, and responses to injury, including energy dissipation and gene expression. These unique characteristics may explain differences in OA prevalence and suggest differences in susceptibility to PTOA.Understanding differences in the response to injury and potential susceptibility to OA can lead to the development of preventative or treatment strategies.Gene expression, cartilage injury, chondrocyte, multiphoton microscopy, cartilage biomechanical properties, PTOA.The prevalence of OA is variable among joints; however, most laboratory studies are performed on a single joint - most commonly the knee, and extrapolated to other joints such as the ankle or shoulder. A small number of studies have compared knee and ankle cartilage and reported differences in mechanical properties and gene expression.There are differences in baseline cell density and gene expression, and differences in response to injury, including gene expression and cell death. This suggests that there are inherent differences leading to varying susceptibilities in OA prevalence among joints. Joint-specific treatments may improve OA therapies.

Project description:BackgroundIn a previous report, we have identified the cannabinoid receptor 2 (CB2) agonist HU308 to possess a beneficial effect in preventing age and trauma-induced osteoarthritis (OA) in mice. The effects of HU308 were largely related to the capacity of this compound to induce cartilage anabolism which was dependent on the CREB/SOX9 axis, and exhibited pro-survival and pro-proliferative hallmarks of articular cartilage following treatment. Here, we utilized the novel cannabinoid-fenchone CB2 agonists (1B, 1D), which were previously reported to render anti-inflammatory effects in a zymosan model.MethodsInitially, we assessed the selectivity of CB2 using a Gs-protein receptor cAMP potency assay, which was also validated for antagonistic effects dependent on the Gi-protein receptor cAMP pathway. Based on EC50 values, 1D was selected for a zymosan inflammatory pain model. Next, 1D was administered in two doses intra-articularly (IA), in a post-traumatic medial meniscal tear (MMT, Lewis rats) model, and compared to sham, vehicle, and a positive control consisting of fibroblast growth factor 18 (FGF18) administration. The histopathological assessment was carried out according to the Osteoarthritis Research Society International (OARSI) guidelines for rat models following 28 days post-MMT.ResultsThe G protein receptor assays confirmed that both 1B and 1D possess CB2 agonistic effects in cell lines and in chondrocytes. Co-administering a CB2 antagonists to 25 mg/kg 1D in a paw inflammatory pain model abolished 1D-related anti-swelling effect and partially abolishing its analgesic effects. Using an MMT model, the high dose (i.e., 24 µg) of 1D administered via IA route, exhibited reduced cartilage damage. Particularly, this dose of 1D exhibited a 30% improvement in cartilage degeneration (zonal/total tibial scores) and lesion depth ratios (44%), comparable to the FGF18 positive control. Synovitis scores remained unaffected and histopathologic evaluation of subchondral bone damage did not suggest that 1D treatment changed the load-bearing ability of the rats. Contrary to the anabolic effect of FGF18, synovial inflammation was observed and was accompanied by increased osteophyte size.ConclusionThe structural histopathological analysis supports a disease-modifying effect of IA-administered 1D compound without any deleterious effects on the joint structure.

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Inhibition of early response genes prevents changes in global joint metabolomic profiles in mouse post-traumatic osteoarthritis.

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