Identification of a novel regulator of adamts-5-mediated aggrecan degradation in chondrocytes

Collins, I., Yamamoto, K., Troeberg, L. and Wann, A.K. (2021) Identification of a novel regulator of adamts-5-mediated aggrecan degradation in chondrocytes. Osteoarthritis and Cartilage, 29 (Supplement 1), S100-S100. (doi:10.1016/j.joca.2021.02.137).

Abstract

Purpose: there are currently no therapies to reduce cartilage loss in osteoarthritis. Our group has been studying the molecular mechanisms regulating the activity of proteases involved in osteoarthritis pathogenesis to identify new therapeutic targets. To do this, we have focused on proteins required for formation and function of an organelle called the primary cilium. We have previously seen that a hypomorphic mutation affecting the ciliary protein IFT88 resulted in elevated matrix catabolism in vitro, which was possibly due to disrupted regulation of protease activity by LRP-1-mediated endocytosis. Here, we aimed to identify other ciliary proteins that regulate proteolytic matrix degradation, determine the mechanism by which these proteins may be acting, and investigate whether expression of the genes encoding these proteins is altered in osteoarthritis.

Methods: proteins with different roles in cilia formation and function were depleted in a mouse chondrocyte line using siRNAs. Proteolytic matrix degradation was measured by culturing cells with purified aggrecan and subsequently detecting the neoepitope AGEG on aggrecan fragments by western blot. qPCR, western blotting and microscopy were used to investigate mechanisms of protease activity regulation.

Results: siRNA-mediated knockdown of the ciliary kinase TTBK2 resulted in increased AGEG-positive aggrecan fragment production relative to control cells treated with non-targeting siRNA. Elevated AGEG in TTBK2 knockdown cells was abolished with combined knockdown of TTBK2 and ADAMTS-5. TTBK2 knockdown was associated with a 20% increase in the expression of Adamts5 mRNA. Expression of targets of the Hedgehog signalling pathway, which is dependent on the cilium and has previously been linked to regulation of cartilage matrix degradation, was not affected in TTBK2 knockdown cells. Activation of the Hedgehog pathway with recombinant Indian Hedgehog did not affect AGEG production. Extracellular levels of the endogenous protease inhibitor TIMP-3 were unaffected by TTBK2 knockdown.

Conclusions: these Results indicate that TTBK2 is a novel regulator of ADAMTS-5-mediated aggrecan degradation in chondrocytes. The mechanism by which TTBK2 regulates ADAMTS-5 activity likely does not involve Hedgehog signalling or TIMP-3, and work is ongoing to determine the contribution of transcriptional changes to elevated ADAMTS-5 activity. Current studies are also addressing whether TTBK2 regulates ADAMTS-5 endocytosis or its extracellular activation. Further investigation of TTBK2 in more clinically relevant contexts is now required, particularly as exploration of global gene expression datasets from RNA-sequencing in normal and osteoarthritic knee cartilage showed that TTBK2 expression is statistically significantly downregulated in osteoarthritis.

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Contributors

Author: A.K. Wann

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