The regulatory effects of stromal interactional molecule-2 on Ca2+ release activated Ca2+ channel of chondrocytes

Abstract

Objective To investigate the regulatory effects of stromal interaction molecule-2 (STIM2) on Ca2+ release activated Ca2+ (CRAC) channel of chondrocytes using STIM2 overexpression model. Methods Chondrocytes were harvested from degenerative (osteoarthritic chondrocytes) or normal regions (normal chondrocytes) of articular cartilage during total knee arthroplasty. CRAC channel function of chondrocytes was tested with Real-time calcium imaging. The protein expression level of STIM2 was examined with Western Blot. The STIM2 overexpression plasmid was constructed and transfected into chondrocytes. The transfect efficiency was verified by cell fluorescence, Real-time PCR, and Western Blot. Results Compared with normal chondrocytes (2.87±0.32 folds, 231.96±17.82 s), osteoarthritic chondrocytes (2.13±0.12 folds, 336.85±15.88 s) demonstrated lower peak intensity of calcium signal in the presence of thapsigargin, and required longer duration to reach saturated calcium concentration after exogenous calcium addition. STIM2 expression level was increased in osteoarthritic chondrocytes. After plasmid transfection, the fluorescence intensity, gene expression and protein level of STIM2 in chondrocytes were increased. Compared with chondrocytes in control group (4.58±0.28 folds, 4.34±0.33 folds), chondrocytes of STIM2 overexpression group (2.60±0.19 folds, 2.24±0.19 folds) showed decreased calcium signal peak intensity upon either thapsigargin stimulation or exogenous calcium addition. Conclusion STIM2 negatively regulates CRAC channel function and decreases stored-Ca2+ in chondrocytes. Key words: Osteoarthritis; Chondrocytes; Calmodulin; Calcium channels