Background/Purpose: Transient receptor potential channel 1 (TRPC1) is a widely expressed mechanosensitive ion channel located within the endoplasmic reticulum membrane. It is crucial for the refilling of depleted internal calcium stores as part of calcium-dependent signaling. Considering the impact of calcium signaling regulation on both homeostatic and inflammatory signaling in chondrocytes, we aimed to examine the role of TRPC1 within cartilage homeostasis and osteoarthritis by monitoring expression during human and murine osteoarthritis (OA) and by assessing the impact of TRPC1 deficiency on OA development and chondrocyte phenotypic stability.

Methods: 10-week old male WT and Trpc1^-/- mice were subjected to destabilization of the medial meniscus (DMM) and were analyzed by microCT, RNA sequencing, histology and immunofluorescence staining at either 2 weeks or 8 weeks post-surgery for OA severity, TRPC1 expression and phenotypic marker expression. TRPC1 protein expression was evaluated in human OA cartilage samples by immunohistochemistry. In vitro, the phenotype of knee articular chondrocytes obtained from 4 day old mice was compared by QPCR, Western blot, SA-b-Gal staining and immunofluorescence. Intracellular calcium mobilization was measured by fluo-4 calcium influx assay.

Results: TRPC1 was found to be depleted from chondrocytes during both human and murine OA development. Trpc1^-/- mice subjected to DMM developed a more severe OA-like phenotype than wild type (WT) controls. Analysis 2 weeks post-DMM revealed an increased rate of chondrocyte survival in Trpc1^-/- cartilage, with remaining cells expressing less SOX9 and increased levels of MMP13 and the senescence marker p16^INK4a. RNA sequencing followed by GO analysis of articular cartilage from Trpc1-/- and WT mice identified a set of differentially expressed genes related to cell number, apoptosis and extracellular matrix organization biological processes.In vitro, stimulation of WT chondrocytes with bFGF, ionomycin or thapsigargin all led to an increase in intracellular calcium, however these effects were significantly reduced in Trpc1^-/-chondrocytes. Although Trpc1^-/- chondrocytes expressed similar levels of chondrocyte differentiation markers at basal level as WT controls, Trpc1^-/- chondrocytes lost expression of type II collagen and increased type I collagen expression significantly faster than WT during serial passage. Replicative senescence and p16^INK4a expression was found to be significantly higher in Trpc1^-/-, both during passage and following IL1 stimulation.

Conclusion: Trpc1^-/- chondrocytes were less able to maintain a stable chondrocyte phenotype in vitro than WT, indicating that TRPC1 activity is required for chondrocyte phenotypic stability. TRPC1 loss was associated with early stages of OA development, while Trpc1^-/- mice developed a more severe OA-like phenotype following DMM surgery. Both in vivo following DMM, andin vitro during passage and following IL1 stimulation, Trpc1^-/- chondrocytes demonstrated an increased susceptibility to cellular senescence. These findings suggest that TRPC1 is a protective factor required for cartilage homeostasis during conditions of physiological challenge.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/loss-of-transient-receptor-potential-channel-1-trpc1-links-regulation-of-intracellular-calcium-to-cellular-senescence-and-leads-to-development-of-osteoarthritis/