Background/Purpose:

Basic calcium phosphate crystals (BCP) are frequently found in osteoarthritic joints. Whether BCP crystal deposition is a cause or consequence of osteoarthritis (OA) pathology remains contentious. In OA, chondrocytes undergo a phenotype shift resulting in increased production of cartilage degrading enzymes (particularly matrix metalloproteinase 13, MMP13) and production of abnormal extracellular matrix components (eg type X collagen, COL10), disrupting cartilage integrity and leading to cartilage loss. The purpose of this study was to determine whether BCP crystals can induce the OA-associated phenotype change in chondrocytes.

Methods:

Primary human chondrocytes were exposed to 50µg/ml BCP crystals (equivalent to the concentration present in synovial fluid in patients with OA) for up to 48h. Chondrocyte phenotypic marker expression (SOX9, RUNX2, IHH, MMP13, COL10A1) was measured by RT-qPCR, ELISA and immunocytochemistry. BCP crystal-induced signalling pathway activation was identified using western blotting, pharmacological inhibitors and RNA interference. Intracellular calcium levels were measured using the calcium-sensitive dye, Fluo4.

Results: Exposure to BCP crystals resulted in changes in chondrocyte phenotypic markers indicating cells were adopting a more OA-like phenotype. Expression of SOX9 was lower, and RUNX2, IHH and MMP13 higher, in BCP crystal-exposed cells compared to untreated controls. COL10 (a late-stage marker of the osteoarthritic chondrocyte phenotype) was detected at both the RNA and protein level in BCP crystal-exposed but not in untreated cells. BCP crystal exposure resulted in a rapid increase in intracellular calcium levels that was abolished by co-treatment with an antagonist preventing the release of calcium from intracellular stores. The BCP-induced increase in intracellular calcium was dependent on free Ca²⁺ in the extracellular environment and activity of the extracellular calcium sensing receptor (CaSR, a receptor for calcium first messenger signalling and activator of calcium release from intracellular stores). Exposure to BCP crystals resulted in activation of the calcium-sensitive enzyme, calcium/calmodulin kinase 2 (CaMK2) by a mechanism dependent on CaSR activity and the release of calcium from intracellular stores. Inhibition of CaMK2 activity or knockdown of either of the CaMK2 isoforms expressed by human chondrocytes (δ and γ) mitigated the ability of BCP crystals to induce OA-associated changes in chondrocyte phenotype.

Conclusion: BCP crystal exposure results in OA-like changes in chondrocyte phenotype by CaSR-mediated activation of CaMK2. Increased CaMK2 activity has been observed in osteoarthritis and is implicated in the disease-associated change in chondrocyte phenotype. Our data indicate the presence of BCP crystals within osteoarthritic joints may contribute to the increased CaMK2 activation seen in disease and may promote OA progression by inducing disease-associated changes in chondrocyte phenotype.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/basic-calcium-phosphate-crystals-induce-osteoarthritis-associated-changes-in-chondrocyte-phenotype-through-activation-of-calcium-calmodulin-kinase-2/