Role of Ciliary Protein Intraflagellar Transport Protein 88 in the Regulation of Cartilage Thickness and Osteoarthritis Development in Mice

INTRODUCTION

Articular cartilage absorbs and transmits mechanical loads generated by muscle contraction and weight-bearing during physical activity. Cartilage can be broadly divided into a noncalcified zone and a calcified zone adjacent to bone. Total cartilage thickness is allometrically scaled to the size of the organism, ensuring that chondrocytes experience similar force irrespective of the mass of the individual animal (1). Articular cartilage is extremely mechanosensitive, with chondrocytes closely monitoring and remodeling the extracellular matrix in response to physiologic loading (2, 3). Physiologic mechanics are critical for cartilage development and homeostasis (4), since their loss has been demonstrated to lead to cartilage thinning (atrophy) (5, 6).

Pathologic, supraphysiologic mechanical loading leads to the development of osteoarthritis (OA), in which degradation of the cartilage occurs, resulting in a loss of integrity of the articular surface (6). Cellular responses to mechanical force include the release of fibroblast growth factor receptor type 2 and transforming growth factor β (TGFβ) from the matrix upon application of a mechanical load, as well as activation of a hedgehog (Hh) ligand, Indian hedgehog (7-10). A number of other pathways are implicated in cellular mechanotransduction, including connexin and ion channel opening and integrin activation. It is not yet understood how chondrocytes in cartilage integrate these cues as the cartilage matures through adolescence and as adaptation occurs to prepare for lifelong loading.

As in most cells in the body, articular chondrocytes express a single immotile primary cilium (11), a microtubule-based organelle reliant on intraflagellar transport (IFT) proteins, including IFT88. Components of the Hh pathway localize to the cilium, supporting bidirectional modulation of signaling (12). Cilia have been directly linked with other growth factor–signaling pathways such as TGFβ signaling, and have also been indirectly linked with a large, growing list of signaling pathways (13), many of which are pertinent to cartilage health. The primary cilium has also been implicated as a “mechanosensory” structure (14). Findings from in vitro experiments in chondrocytes have implied that ciliary protein IFT88 is associated with both compression-induced production of extracellular matrix proteins (15) and impaired clearance of aggrecanases, resulting in exacerbated aggrecan degradation (16).

Developmental mutations in ciliary genes, including Ift88, result in impaired embryonic patterning as a result of disrupted Hh signaling (17). Cartilage-specific deletion of Ift88 results in disrupted long bone and articular cartilage formation (18). Cartilage-specific deletion of Ift80 in mice in the first 2 weeks of postnatal life was found to result in thickening of the articular cartilage (19). However, despite its putative influence over a range of processes that regulate health and disease in cartilage, we have very limited direct knowledge regarding the postdevelopmental influence of ciliary IFT. We hypothesized that in a mouse model, ciliary protein IFT88 plays a crucial role in mediating cartilage homeostasis in adult animals.

DISCUSSION

In the current study, we explored the influence of ciliary protein IFT88 in postnatal mouse articular cartilage in vivo, by depleting its expression in chondrocytes at different stages of postnatal skeletal maturation. When Ift88 was deleted in the mice at 4, 6, and 8 weeks of age, we observed rapid cartilage thinning, largely in the calcified cartilage within the medial joint compartment. Thinning was determined to be indicative of atrophy rather than degeneration, since there was no disruption of the articular surface. However, as mice aged, cartilage atrophy was associated with increased severity of spontaneous OA and DMM-induced OA.

We speculate that the Ift88-dependent effects in the thicker medial compartment of the mouse joint are mechanically driven (6) in a manner analogous to the bone “mechanostat” proposed by Frost in 1987 (31). In essence, this model ensures that chondrocytes mechanoadapt the extracellular matrix so as to experience force within a narrow window (1). Since cartilage thickness can be restored in Ift88–conditional KO mice with wheel exercise, this suggests that ciliary protein IFT88 may influence, but is not solely responsible for, cartilage mechanoadaptation in vivo. Similar modes of action have been proposed in the context of epithelial response to renal flow (14, 32). Our findings contrast with the observation that in mice, deletion of Ift88 during development leads to increased cartilage thickness (17), possibly indicating a changing influence of Ift88 with skeletal maturation.

Since atrophy in Ift88–conditional KO mice is largely restricted to calcified cartilage, we speculate that this represents a failure of cartilage hypertrophy during maturation, in a load-dependent manner. Cartilage thinning was not associated with increased matrix catabolism, enhanced subchondral bone thickness, osteoclast activity, or density changes (BV/TV) in the epiphysis, but we cannot exclude the possibility that calcified cartilage is transitioning to the bone in these mice. Calcification of mouse cartilage has recently been linked to Enpp1, a pyrophosphatase believed to inhibit calcification through Hh signaling (30). Findings from our molecular analyses indicated that Enpp1 positively correlates with Ift88 expression, indicating a reduction in the levels of an inhibitor of calcification in Ift88–conditional KO mice. This could result in accelerated ossification analogous to that seen in the growth plates of ciliary protein mutant mice and in articular cartilage upon postnatal activation of Hh (25).

Prior studies investigating congenital mutations (33) or constitutive deletions of Ift88 (17, 18, 34) demonstrated that Ift88 plays a role in mouse limb and joint development. Mouse models targeting other ciliary components, Kif3a, Bardet-Biedl syndrome proteins, and Ift80, also implicate ciliary machinery in musculoskeletal development (19, 28). This influence over skeletal development is also exemplified by the human skeletal ciliopathies (35, 36). The most important molecular pathway associated with ciliopathy is Hh, although other pathways have also been described (10, 37-41). Hh signaling largely switches off in adulthood (42) but is reactivated in OA (8). We investigated the molecular basis of Ift88-dependent cartilage atrophy by identifying the correlation between Ift88 expression (reflecting efficiency of deletion) and 44 molecules previously demonstrated to be associated with ciliary signaling and cartilage biology. In addition, we explored Hh signaling by directly visualizing Gli1 expression in murine cartilage using RNAScope. The gene that correlated most strongly with ciliary signaling was transcription factor Tcf7l2, previously shown to influence and interact with Hh and β-catenin signaling pathways in cartilage (37). Other genes whose levels correlated with Ift88 included Gli2, Ctgf, and Enpp1, although these correlations were only statistically significant before Bonferroni correction.

While a gene expression analysis of microdissected mouse cartilage did not show a correlation between Ift88 and classic Hh pathway molecules (Gli1, Ptch1), an individual cell analysis using RNAScope revealed increased levels of Gli1 expression in aggrecanCreERT2;Ift88fl/fl mouse chondrocytes, suggesting a reciprocal relationship between Hh signaling and Ift88. Therefore, we propose that loss of Ift88 disrupts ciliary-mediated repression of Hh signaling, resulting in net increases in Gli1 expression. This is consistent with findings from previous studies in constitutive Ift88 deletion (18) and endochondromas (43). In this model, our observation that cartilage atrophy was rescued and basal Gli1 expression levels were normalized in aggrecanCreERT2;Ift88fl/fl mice following wheel exercise provides critical evidence of a link with mechanical loading. Our data imply that in postnatal mouse articular cartilage, ciliary protein IFT88 safeguards the progressive mechanoadaptation of adolescent mouse cartilage, supporting the creation and maturation of fit-for-purpose adult mouse articular cartilage by ensuring appropriate levels of Hh signaling.

As previously described (44), Ift88 was deleted in mouse chondrocytes using induction of Cre recombinase expression on the aggrecan promotor, which ensured sufficient expression in mice from adolescence through adulthood. Despite the use of this method, we observed only a 40% reduction in Ift88-positive chondrocytes in the tibial articular cartilage of aggrecanCreERT2;Ift88fl/fl mice. Observations in the tdTomato reporter line of mice indicated that Ift88 deletion occurred in only a small proportion of chondrocytes, but this was not exclusive to any knee compartment, and therefore it is unlikely that this finding could be attributed to differences between the medial and lateral sides of the knee joint. We also recognize that, due to the challenges associated with imaging cilia in cartilage, we have not yet been able to address the question of whether primary cilia would be negatively impacted as a result of Ift88 deletion. Thus, we cannot conclude that molecular changes are a direct consequence of the loss of cilia. To date, we have not yet conducted experiments inducing Ift88 deletion in mice older than age 8 weeks to evaluate whether this gene is as influential later in adulthood, or assessed how behavior of the aggrecanCreERT2;Ift88fl/fl mice could be altered.

In summary, these data demonstrate that IFT88 is highly influential in adolescent mouse and adult mouse articular cartilage as a positive regulator of cartilage thickness, guiding cartilage calcification during maturation and safeguarding physiologic Hh signaling in adult mouse cartilage in response to mechanical cues.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Coveney had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design

Coveney, Wann.

Acquisition of data

Coveney, Zhu, Miotla-Zarebska, Chang, McSorley.

Analysis and interpretation of data

Coveney, Stott, Parisi, Batchelor, Duarte, Vincent, Wann.

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