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

Ciliary IFT88 protects coordinated adolescent growth plate ossification from disruptive physiological mechanical forces

Compared with our understanding of endochondral ossification, much less is known about the coordinated arrest of growth defined by the narrowing and fusion of the cartilaginous growth plate. Throughout the musculoskeletal system, appropriate cell and tissue responses to mechanical force delineate morphogenesis and ensure lifelong health. It remains unclear how mechanical cues are integrated into many biological programs, including those coordinating the ossification of the adolescent growth plate at the cessation of growth. Primary cilia are microtubule-based organelles tuning a range of cell activities, including signaling cascades activated or modulated by extracellular biophysical cues. Cilia have been proposed to directly facilitate cell mechanotransduction. To explore the influence of primary cilia in the mouse adolescent limb, we conditionally targeted the ciliary gene Intraflagellar transport protein 88 (Ift88fl/fl) in the juvenile and adolescent skeleton using a cartilage-specific, inducible Cre (AggrecanCreERT2 Ift88fl/fl). Deletion of IFT88 in cartilage, which reduced ciliation in the growth plate, disrupted chondrocyte differentiation, cartilage resorption, and mineralization. These effects were largely restricted to peripheral tibial regions beneath the load-bearing compartments of the knee. These regions were typified by an enlarged population of hypertrophic chondrocytes. Although normal patterns of hedgehog signaling were maintained, targeting IFT88 inhibited hypertrophic chondrocyte VEGF expression and downstream vascular recruitment, osteoclastic activity, and the replacement of cartilage with bone. In control mice, increases to physiological loading also impair ossification in the peripheral growth plate, mimicking the effects of IFT88 deletion. Limb immobilization inhibited changes to VEGF expression and epiphyseal morphology in Ift88cKO mice, indicating the effects of depletion of IFT88 in the adolescent growth plate are mechano-dependent. We propose that during this pivotal phase in adolescent skeletal maturation, ciliary IFT88 protects uniform, coordinated ossification of the growth plate from an otherwise disruptive heterogeneity of physiological mechanical forces

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

The ciliary protein intraflagellar transport 88 is required for the maturation, homeostasis and mechanoadaptation of articular cartilage

Purpose: The development, maturation and maintenance of articular cartilage depends on the integration of external cues, such as mechanics, with intrinsic cell signalling programmes, such as hedgehog (Hh) signalling. Aberrant mechanics and the post-natal activation of Hh signalling have both been associated with the development of murine and human OA. It remains to be fully elucidated how chondrocytes transduce and integrate these cues in vivo. Chondrocytes assemble a primary cilium, a microtubule-based organelle with a devoted trafficking machinery, IntraFlagellar Transport or IFT. In vitro studies indicate ciliary IFT helps tune the chondrocyte response to Hh ligand and the anabolic matrix response to cell compression. Recently, we have shown loss of function of IFT88, inhibiting ciliogenesis in chondrocytes, impairs LRP-1 mediated endocytotic clearance of proteases, resulting in increased aggrecanolysis. While constitutive and peri-natal disruption of ciliary proteins, Hh signalling and altered mechanics, all drastically alter joint development in vivo, the influence of IFT in adult cartilage homeostasis remains unknown.Methods: IFT88 was targeted using a cartilage-specific, inducible mouse line (ACANCreERT2;IFT88fl/fl : cKO hereafter). Cre activity was validated by qPCR, and using a ROSA26tdtomato reporter line. IFT88fl/fl mice, also receiving I.P injections of tamoxifen, were used as controls. Tibial articular cartilage was assessed 2, 14 or 26 weeks post tamoxifen, at 8, 10, 22 and 34 weeks of age respectively, using histomorphometric analyses, including measurements of articular cartilage thickness, relative calcification, and OARSI score and by means of immunohistochemistry (IHC). The DMM model, which destabilising the joint, was performed at 10 weeks of age. Means ± S.D are quoted throughout, Mann-Whitney U-tests used for statistical comparisons.Results: The Tdtomato reporter demonstrated ACANcre activity in hip and throughout knee cartilage. Tamoxifen treatment of cKO mice resulted in a 50% reduction of IFT88 mRNA in articular cartilage (p=0.02, n=6 ctrl, 14 cKO). In control mice, tibial articular cartilage thickens between 8 and 22 weeks of age, most notably on the medial plateau. Calcified cartilage (below the tidemark) progressively increases on both plateaus, between 6 and 22 weeks of age. IFT88 depletion (cKO) resulted in thinner medial articular cartilage (MC), compared with controls, at all 5 time-points. Fig 1.A shows MC thickness in adult control (crosses) and cKO (circles). In control mice, MC thickness increased from 99.0 +/- 9.2 μm at 8 weeks of age to 108.9 +/- 7.2 μm at 10 weeks of age. Tamoxifen treatment, at 8 weeks of age, inhibited this increase in cKO mice (MC thickness at 10 weeks was 96.2 +/- 7.4 μm, p=0.02, compared with 10 week ctrl, n=7). By 22 weeks of age mean MC thickness in cKO was 90.2 +/- 3.3 μm compared with 111.6 +/- 10.1 μm in control animals (p=0.0002, n= 7 and 10 respectively). By 34 weeks MC had continued to thin, but this was now associated with surface damage and osteophyte formation. In the most extreme case, MC was completely lost (Fig.1B). In contrast, lateral plateau thickness and OARSI score was unaffected. At all time-points thinning was attributable to loss of calcified cartilage. IHC analyses revealed no striking differences in collagen X expression, NITEGE neoepitope staining or the expression of LRP-1β. 12 weeks post DMM, OARSI scores were statistically significantly higher in cKO mice.Conclusions: Progressive thickening and calcification in the mouse medial compartment illustrates the continued adaptation of adolescent and adult articular cartilage, in the medial loading environment. IFT88 deletion inhibits MC thickening, leading to atrophy, which then predisposes the joint to spontaneous OA as the mouse ages. The lateral compartment is relatively unaffected. We propose this may be due, in part, to disruption of mechanotransduction and downstream anabolic remodelling in medial cartilage. Deletion of IFT88 impairs the progressive calcification of articular cartilage, in both compartments, which may be due to disruption of intrinsic cartilage Hh signalling. Mechanistic experiments, dissecting the relative roles and integration of IFT, mechanics and Hh in the context of adult cartilage are on-going. We conclude that IFT88 maintains a profound influence in post-natal articular cartilage homeostasis and protection from OA.</div

The ciliary protein intraflagellar transport 88 is required for the maturation, homeostasis and mechanoadaptation of articular cartilage

Purpose: The integration of external cues, such as mechanics, with intrinsic cell signalling programmes, such as hedgehog (Hh) signalling, is crucial for the development, maturation and homeostasis of articular cartilage. Activation of Hh signalling in adulthood and pathophysiological mechanics, have both been associated with the development of murine and human OA. But, how chondrocytes might transduce and integrate these cues remains unknown. A microtubule-based organelle, the primary cilium, most noted for its crucial role in Hh signalling, is assembled by chondrocytes and possesses a devoted trafficking machinery, IntraFlagellar Transport or IFT. In vitro studies indicate chondrocyte helps tune the anabolic matrix response to compression and the response to Hh ligand. In vivo, the primary cilium has been proposed to be a platform for the integration of mechanics and Hh signalling in musculoskeletal tissues. While constitutive and peri-natal disruption of ciliary proteins, Hh signalling and altered mechanics, all drastically alter joint development in vivo, the influence of IFT in adult cartilage homeostasis remains unknown.Methods: IFT88 was targeted using a cartilage-specific, inducible mouse line (ACANCreERT2;Ift88fl/fl : cKO hereafter). Cre activity was validated by qPCR, RNA scope and a ROSA26tdtomato reporter line. Ift88fl/fl mice, also receiving I.P injections of tamoxifen, were used as controls. Tibial articular cartilage was assessed 2, 14 or 26 weeks-post tamoxifen, at 8, 10, 22 and 34 weeks of age respectively, using histomorphometric analyses, including measurements of articular cartilage thickness, relative calcification, subchondral bone thickness, and OARSI score by means of immunohistochemistry (IHC). The surgical DMM model, which destabilises the joint, was performed at 10 weeks of age. To explore the role of physiological mechanics, mice were allowed two weeks of voluntary wheel exercise immediately following tamoxifen administration at 8 weeks of age. qPCR was performed on micro dissected articular cartilage at 10 weeks of age in control and cKO. RNAscope was performed on cryosections of articular cartilage from 10 week old mice control and cKO. Means ± S.D are quoted throughout, Mann-Whitney U-test or Fisher’s test were used for statistical comparisons.Results: In our previous OARSI abstract of 2020 we described the phenotype arising in IFT88 cKO mice. Here we outline this in further detail and with an exploration of underlying mechanisms. Tamoxifen treatment of cKO mice resulted in a 50% reduction of Ift88 mRNA in articular cartilage (p=0.02, n=6 control, 14 cKO). Ift88 (cKO) mice had thinner medial articular cartilage (MAC), compared with controls, at all 5 time-points (Fig 1.A) In control mice, MAC thickness increased from 102.57μm (95% CI [94.30, 119.80]) at 8 weeks of age to 108.68 +/- (95% CI [101.32, 116.42]) at 10 weeks of age. Tamoxifen treatment, at 8 weeks of age, inhibited this increase in cKO mice (MC thickness at 10 weeks was 96.20 μm (95% CI [90.04, 102.36]), p=0.02, compared with 10 week ctrl, n=7). By 22 weeks of age mean MAC thickness in cKO was 90.16μm (95% CI [87.11, 93.22]) compared with 111.60μm (95% CI [104.34, 118.79]) in control animals (p=0.0002, n= 7 and 10 respectively). By 34 weeks MAC had continued to thin to 84.55μm (95% CI [75.43, 93.67]) in cKO, but this was now associated with surface damage and osteophyte formation. In the most extreme case, MAC was completely lost (Fig.1B). In contrast, lateral plateau thickness and OARSI score were unaffected. Calcified cartilage (below the tidemark) progressively increases on both plateaus, between 6 and 22 weeks of age and at all time-points thinning was attributable to the relative loss of calcified cartilage implying a failure of calcification. IHC analyses revealed no striking differences in collagen X expression, NITEGE neoepitope. There were no measurable increases in subchondral bone thickness or changes in osteoclastic activity in cKO mice. 12 weeks post DMM, OARSI scores were statistically significantly higher in cKO (29.83 +/- 7.69) than control (22.08 +/- 9.30, p&lt; 0.05, n= 15 both groups). Two weeks of voluntary wheel exercise rescued cartilage atrophy in cKO mice (p&lt; 0.0001), whilst no change was observed in controls. RNA isolated from microdissected articular cartilage of 10 week old control and cKO mice, two weeks post tamoxifen, revealed a statistically significant correlation between Ift88 and Tcf7l2 expression after Bonferroni correction (p=0.026). Ctgf, Gli2 and Enpp1 were also positively correlated with Ift88 expression before correction (p=0.002, p=0.0037, and p=0.009 respectively). RNA scope analysis of AC found a statistically significant (p&lt; 0.0001, n=4 in both groups) decrease in Ift88 positive cells in cKO (27.78%) compared with controls (45.18%), whilst also showing an increase in Gli1 positive cells in cKO (50.42%) compared with controls (23.63%) (p&lt; 0.0001, n=4 in both groups).Conclusions: Progressive thickening and calcification in the mouse medial compartment illustrates the continued mechanoadaptation of adolescent and adult articular cartilage. Depletion of the ciliary gene Ift88 inhibits medial articular cartilage thickening, leading to atrophy, which then predisposes the joint to spontaneous OA. The lateral compartment is relatively unaffected. We propose this may be due, in part, to disruption of mechanotransduction and downstream anabolic remodelling in medial cartilage. Deletion of Ift88 impairs the progressive calcification of articular cartilage, in both compartments, which may be due to disruption of Hh signalling, which is also mechanosensitive. Ift88 expression was correlated with Tcf7l2, previously shown to interact and influence Hh signalling pathways in cartilage. On-going experiments are aiming to dissect the relative roles of IFT, mechanics and Hh in the context of adult cartilage. We conclude that Ift88 is crucial to post-natal articular cartilage homeostasis and chondroprotective against OA.</div