The primary cilia protein IFT88, exerts a profound and evolving influence in the post-natal joint, through joint maturation, adult life and in disease

Purpose: Mechanical forces are critical for joint development, homeostasis and disease. During skeletal development mechanics contributes to formation of the joint, endochondral ossification during long bone elongation and growth plate closure. Physiological loading during adult life underpins articular cartilage health, continuity and thickness. Pathological loading of the joint leads to cartilage degradation and osteoarthritis (OA). However, we don’t yet understand the relative contributions of the cartilage, bone and synovium or cells vs matrix in healthy or pathological mechanotransduction. In both chondrocytes and osteocytes, proteins critical to the assembly and function of the primary cilium have been shown to be important to the response to mechanics. Recently we have shown they regulate cartilage turnover. The cilium is a microtubule-based organelle most famously associated with transduction of extracellular cues including the hedgehog ligand and mechanics. As such mutations to ciliary proteins results in a group of human congenital diseases known as ciliopathies including skeletal disorders, further emphasising the cilium’s critical role in musculoskeletal development. However, little is known about the cilium’s post-natal roles within the joint. A core component of the cilium is IFT88, disruption of which alters mechanically induced matrix production and catabolism in chondrocytes. We hypothesise that the crucial role of the cilium and associated machinery persists into adulthood, is important for the joints response to physiological loading and by extension, cartilage health.Methods: We have created an adult, cartilage-specific, inducible deletion model of IFT88 (ACAN;IFT88fl/flCreERT2) and a pan tissue (ROSA26;IFT88fl/flCreERT2). These were validated by qPCR, western blot, in vitro and a ROSA26tdtomato reporter. Phenotyping of this mouse was conducted using histology, MicroCT and imageJ. The surgical destabilisation of the medial meniscus model (DMM) was used to challenge the joint with aberrant mechanics to induce OA followed by double-blinded OARSI scoring.Results: The reporter mouse reveals that aggrecan cre is driving in the articular surface, growth plate, menisci and some populations within the bone. qPCR indicates an approximate halving of IFT88 mRNA in the ACAN;IFT88fl/flCreERT2, termed cKO hereafter. Activation of the ROSA26;IFT88fl/flCreERT2 also results in a halving of IFT88 mRNA in multiple tissues and reduction of protein in cartilaginous tissue. Between 4 and 10 weeks of age in mice, endochondral ossification is nearing completion and growth plate closure begins. Deletion of IFT88 at 4 weeks of age results in a longer growth plate including enlarged bi-lateral growth plate cartilage at 6 weeks. Deletion at 6 weeks also results in a longer growth plate at 8 weeks; in each case the cKO growth plate remains at the length it was 2 weeks earlier prior to deletion. Between 8 and 10 weeks of age, cKO of IFT88 also results in longer growth plates as growth plate closure is inhibited. Between 6 and 10 weeks of age the articular surface thickens at a linear rate from ∼85μm to 115μm. In the cKO, this thickening over each 2 week period is inhibited. Two weeks after deletion, cartilage is ∼15μm thinner at every time point in the cKO. Further analysis of histology reveals a reduction in the calcified cartilage region, whereas no change was seen in the non-calcified region. At 12 weeks post-DMM there is a significant difference in OARSI score between with higher scores in the cKO. In older mice (6 months of age) there is a pronounced long bone phenotype with loss of trabeculae and total bone volume.Conclusions: We have successfully depleted chondrocytes of IFT88 using an aggrecan Cre system, however potentially only in a subset of the population. IFT88 maintains influence through adolescence and early adulthood. Our data suggests IFT88/the cilium continues to regulate growth plate dynamics during and beyond developmental stages, potentially regulating both the Hh-PTHrP-Hh feedback loop during endochondral ossification and growth plate closure mechanisms. We propose this is by dysregulation of upstream mechanotransduction. IFT88 maintains influence during articular cartilage maturation and in early adulthood. Upon knock out of IFT88, articular cartilage thickness is reduced and associated with changes to the calcified region. This is in some agreement with changes seen in small models of cartilage atrophy, where the mechanism is not yet understood. On the introduction of pathological mechanical load using DMM, IFT88 deletion leads to exacerbation of articular cartilage degradation, potentially owing to loss of physiological mechanotransduction prior to DMM challenge. On-going work is analysing the effects at earlier time points in disease progression (8 weeks post-DMM) and importantly the naive articular surface during adulthood. Collectively, with our previous in vitro work, we propose IFT88 regulates cartilage turnover and potentially ossification of deep zone articular cartilage. We are now testing these hypotheses, targeting other elements of the ciliome and exploring the molecular mechanisms downstream to IFT88 loss in mature and mechanically challenged cartilage to compare with changes seen in atrophy and OA.</p