Background/Purpose: We have recently demonstrated that ELR+ CXC chemokines signaling via the CXCR2 receptor, are produced by healthy chondrocytes and are retained within the cartilage matrix via interactions with heparan sulfate proteoglycans.  They act to support articular cartilage homeostasis by increasing AKT phosphorylation, thus promoting SOX9 expression, extracellular matrix production and chondrocyte survival, particularly during conditions of physiological challenge, indicated by increased chondrocyte apoptosis and cartilage degradation in CXCR2^-/- mice following destabilization of the medial meniscus.  Importantly, the transient receptor potential channel 6 (TRPC6) mechanosensitive ion channel has been shown to be a specific mediator of CXCR2 driven cell migration via AKT signaling.  This project aims to investigate whether TRPC6 activity is required for chondrocyte phenotypic stability and is involved in the mechanism of CXCR2-mediated cartilage homeostasis.

Methods: Costal chondrocytes were isolated from CXCR2^-/- mice, TRPC6^-/- mice and their wild type littermates and expanded in standard conditions.  Gene expression was assessed using real time RT-PCR.  Sulfated proteoglycan content and mineralization of micromass cultured chondrocytes was measured using Alcian blue or alizarin red 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.  Intracellular calcium was chelated by treating cells with BAPTA-AM prior to receptor activation.  AKT phosphorylation was analyzed using Western blot. Calcium mobilization in chondrocytes was measured using a fura-2 assay.

Results: TRPC6 mRNA was detected in wild type murine chondrocytes.  Chondrocytes lacking either TRPC6 or CXCR2 expressed lower levels of the chondrocyte differentiation markers SOX9 and type II collagen in comparison to wild type.  Culture of chondrocytes in micromass resulted in significantly less sulfated proteoglycan production and increased mineralization by TRPC6^-/- chondrocytes in comparison to chondrocytes obtained from wild type littermates. Activation of CXCR2 in articular chondrocytes resulted in increased intracellular calcium mobilization.  In vitro activation of TRPC6 using hyp9 led to an increased AKT phosphorylation, whereas chelation of intracellular calcium inhibited CXCL6-induced phosphorylation of AKT.  Finally, activation of TRPC6 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 calcium channel activity is required for chondrocyte phenotypic stability.  In vitro TRPC6 activation is sufficient to increase AKT phosphorylation and the expression of key chondrocyte phenotypic markers in murine chondrocytes, indicating that TRPC6 may be an ideal therapeutic target aimed at preventing cartilage degradation during osteoarthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-role-of-trpc6-in-cxcr2-mediated-chondrocyte-phenotypic-stability/