Background/Purpose: The ELR+ CXC chemokine CXCL6 is produced by healthy articular chondrocytes and retained within the cartilage matrix via interactions with heparan sulphate proteoglycans. There, it signals via CXCR2 to promote maintenance of articular cartilage homeostasis, particularly during conditions of physiological challenge, which was indicated by increased cartilage degradation in CXCR2^-/-mice following destabilization of the medial meniscus. The transient receptor potential channel 6 (TRPC6) mechanosensitive ion channel was demonstrated to be a specific mediator of CXCR2 driven cell migration. This project aims to investigate whether TRPC6 activity is required for CXCR2-mediated cartilage homeostasis.

Methods: Costal chondrocytes were isolated from CXCR2^-/- mice, TRPC6^-/-mice and their wild type (WT) littermates and expanded under standard conditions. Chondrocyte TRPC channel and phenotypic gene expression was assessed using real time RT-PCR, Western blot and immunohistochemistry. Sulfated proteoglycan content of chondrocyte micromasses was measured using Alcian blue staining and spectrophotometric quantification. CXCR2 was activated using murine CXCL6 whilst TRPC6 was specifically activated using hyp9, a stabilized derivative of the TRPC6-selective activator hyperforin. Calcium mobilization in chondrocytes was measured using a fura-2 calcium influx assay. AKT phosphorylation was analyzed by Western blot.

Results: TRPC6 mRNA and protein was detected in WT murine chondrocytes. TRPC6^-/- and CXCR2^-/- chondrocytes expressed lower levels of the chondrocyte differentiation markers SOX9 and type II collagen in comparison to WT controls, accompanied by less AKT phosphorylation. TRPC6^-/- chondrocyte micromass cultures produced significantly less sulfated proteoglycans in comparison to WT. CXCL6 treatment of WT chondrocytes resulted in increased intracellular calcium mobilization and AKT phosphorylation, which was not observed in CXCL6 treated TRPC6^-/-cells. In vitro activation of TRPC6 using hyp9 led to increased AKT phosphorylation and resulted in a significant increase in SOX9 and type II collagen mRNA expression, together with a decrease in type X collagen mRNA expression.

Conclusion: TRPC6 is required for chondrocyte phenotypic stability and for CXCL6-induced calcium influx and AKT phosphorylation. In vitro TRPC6 activation is sufficient to increase the key chondrocyte phenotypic marker gene expression in murine chondrocytes, indicating that TRPC6 may be an ideal pharmacological target for reducing the loss of chondrocyte phenotypic stability and cartilage degradation during osteoarthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/maintenance-of-chondrocyte-phenotypic-stability-by-trpc6-calcium-channel-activity/