Exploring and exploiting a ciliome-protease-matrix regulatory axis disturbed in osteoarthritis

Purpose: in osteoarthritis (OA) cartilage homeostasis is disturbed sufficiently such that tissue is degraded and destroyed. A carefully balanced relationship between anabolic and catabolic activity is upset within the complex cartilage matrix environment. Mechanics, at the level of the whole joint, tissue and at the cellular level, is proposed to be a major contributor to this imbalance. During cartilage development, mechanical and biological cues are partially transduced by the primary cilium and/or its associated machinery (ciliome). Learning from its roles in development, we wish to understand the post-developmental influence of the ciliome. We have a particular focus on the interaction between the ciliome and protease activity in OA and have recently discovered a novel ciliome-protease regulatory axis acting through LRP-1. We hypothesise that roles of the ciliome alter into adulthood, but influence is maintained, in part, through a ciliome-endocytosis-protease-matrix continuum that is disturbed in OA.Methods: intraflagellar transport proteins (IFTs) carry cargoes to and within the cilium such as the regulatory GTPase arl13b. IFT88 is a core component of the ciliome, which is also physically and mechanistically associated with the centrioles. We have created a cartilage-specific, adult mouse IFT88 deletion model by crossing IFT88fl/fl x Acan-CreER2. This mouse was phenotyped using histology and μCT. We are also using a double transgenic model (ARL13bmCherry; Centrin 2-GFP) that enables in situ visualisation of the cilia and centrioles in cartilage. Chondrocyte polarity and cilia were analysed in situ following confocal imaging, using ImageJ software. In vitro we measured protease activity in both tissue co-culture and recombinant matrix overlay systems, quantifying production of neoepitopes by chondrocytes.Results: IFT88 exerts strong influence in the mouse joint into adolescence with a less acute, but nevertheless sustained influence into adulthood. This includes a disturbed chondrocyte orientation, and changes to the cartilage matrix profile and the bone beneath. In vitro, IFT88 modulates matrix catabolic activity of chondrocytes through LRP-1-mediated endocytosis. Cartilage has a complex organisation that is disturbed in OA. Chondrocyte polarity, and thus a putative ciliome-LRP-1-matrix axis, is highly organised in relation to this.Conclusions: the ciliome is physiologically relevant in adult joints and potentially has a pathophysiological role in OA, possibly through the regulation of protease activity via an LRP-1-protease axis. Currently, we are investigating the rescue of pathophysiological protease activity in vitro by modulating ciliome and peri-ciliary components. In vivo,we are using ageing and pre-clinical disease models to further understand the physiological and pathophysiological relevance of IFT and the ciliome