Inhibition of Early Response Genes Prevents Changes in Global Joint Metabolomic Profiles in Mouse Post-Traumatic Osteoarthritis

Abstract

Osteoarthritis (OA) is the most common degenerative joint disease, and joint injury increases the risk of OA by 10-fold. Although the injury event 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), the production of inflammatory cytokines, and we hypothesize, changes to the joint metabolome. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Flavopiridol is a potent and selective inhibitor of Cdk9 kinase activity, which prevents the transcriptional activation of early response genes. To model post-traumatic osteoarthritis, we subjected mice to non-invasive ACL-rupture joint injury. Following injury, mice were treated with flavopiridol to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1 hour after injury. We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism and others. Importantly, we found that inhibition of primary response gene activation at the time of injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites, which may offer novel targets for cell-mediated secondary joint damage. Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses, and these data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury.\n\nSignificance StatementJoint injury is an excellent predictor of future osteoarthritis. It is increasingly apparent that the acute cellular responses to injury contribute to the initiation and pathogenesis of OA. Although changes to the joint transcriptome have been extensively studied in the context of joint injury, little is known about changes to small-molecule metabolites. Here we use a non-invasive ACL rupture model of joint injury in mice to identify injury-induced changes to the global metabolomic profiles. In one experimental group we prevented the activation of primary response gene transcription using the Cdk9 inhibitor flavopiridol. Through this comparison, 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.