X. Gong (Chongqing, CN)
Southwest hospital Joint SurgeryPresenter Of 1 Presentation
10.4.6 - The mechanism of spontaneous calcium signaling in chondrocytes and its regulation in cartilaginous matrix metabolism
Abstract
Purpose
To investigate the role of spontaneous [Ca2+]i signaling in cartilaginous ECM metabolism regulation, and its correlation with OA severity.
Methods and Materials
Cartilage explants and primary chondrocytes were isolated from porcine knee joints. In situ calcium imaging was performed in cartilage explants with pharmacological interventions. Primary chondrocytes were used for RT-qPCR, WB, live and dead assay, Alcian blue and immunofluorescence staining. By using Fluo-8 AM based calcium imaging system, cartilage samples at different OA stages collected from patients undergoing knee arthroscopic surgery were used to analyze the spatiotemporal features of spontaneous [Ca2+]i signaling of in situ chondrocytes.
Results
we found that spontaneous [Ca2+]i signaling of in situ porcine chondrocytes was tightly regulated by [Ca2+]o influx, InsP3Rs mediated [Ca2+]i store release, and Orais mediated CRAC activation. [Ca2+]o deprivation was associated with decreased cell viability, and expression levels of ECM deposition genes. Whereas blocking ER Ca2+ release with InsP3R inhibitors significantly up-regulated ECM degradation genes, down-regulated SOX11 expression level, and was accompanied by decreased proteoglycan and collagen type Ⅱintensity. Furthermore, our data showed zonal dependent spontaneous [Ca2+]i signaling in healthy cartilage samples under 4 mM calcium environment.This signal was significantly attenuated in healthy cartilage samples when cultured in calcium free environment. No significant difference was found in [Ca2+]i intensity oscillation in chondrocytes located in middle zones among ICRS 1-3 samples, but OA severity dependent spatiotemporal features of spontaneous [Ca2+]i oscillations of deep zone chondrocytes was observed.
Conclusion
Our data provided evidence that spontaneous [Ca2+]i signaling of in situ porcine chondrocytes was tightly regulatedby [Ca2+]o influx, InsP3Rs mediated [Ca2+]i store release, and Orais mediated CRAC activation. Both [Ca2+]o concentration and InsP3Rs mediated ER Ca2+release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.This might reflect the role of spontaneous [Ca2+]i signaling during OA progression and provide insight into articular cartilage degradation during OA progression.