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