Calreticulin silencing inhibits extracellular matrix synthesis of human gingival fibroblasts cultured on three-dimensional poly(lactic-co-glycolic acid) scaffolds by inhibiting the calcineurin/nuclear factor of activated T cells 3 signalling pathway

Abstract

BackgroundThe retraction and compression of gingival tissue have a significant impact on the efficiency and stability of orthodontic treatment, but the underlying molecular mechanism has not been fully elucidated. The aim of the current study was to investigate the effects of mechanical forces on the expression level of calreticulin (CRT), the activity of the calcineurin (CaN)/nuclear factor of activated T cells (NFAT) 3 signalling pathway, and extracellular matrix (ECM) synthesis in human gingival fibroblasts (HGFs) cultured on three-dimensional (3D) poly(lactic-co-glycolic acid) (PLGA) scaffolds and to further explore the mechanical transduction pathways that may be involved. Materials and methodsA mechanical force of 25 g/cm2 was applied to HGFs for 0, 6, 24, 48, or 72 h. The expression of CRT, CaN, NFAT3, phosphorylated NFAT3 (p-NFAT3) and type I collagen (COL-I) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Subsequently, small interfering RNA (siRNA) was used to knock down the expression of CRT in HGFs, and the impacts of the applied force on the expression levels of CaN, NFAT3, p-NFAT3, and COL-I were also evaluated by RT-qPCR and western blotting. ResultsThe application of mechanical force on HGFs cultured on 3D PLGA scaffolds led to a significant increases in CRT, CaN, and COL-I expression as well as a decrease in p-NFAT3 expression. However, the effects of mechanical force on CaN, p-NFAT3, and COL-I expression were reversed following downregulation of CRT and displayed a significant decrease in CaN/NFAT3 activity and COL-I synthesis. ConclusionThis study showed that the CaN/NFAT3 signalling pathway and CRT appear to be involved in the mechanotransduction of HGFs, and downregulation of CRT inhibits COL-I synthesis potentially via the CaN/NFAT3 signalling pathway. Taken together, these findings ultimately provide novel insight into the mechanisms underlying mechanical force-induced ECM synthesis, which may be conducive to the development of targeted therapeutics to treat fibrotic diseases, including gingival fibrosis caused by orthodontic treatment.