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T. T. Wong, R. Su, L. F. Seet; Induction of Alpha Smooth Muscle Actin Expression in Tenons Fibroblasts Under Mechanically-Induced Stress Involves SPARC and the TGFβ Signalling Pathway. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6107.
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Alpha smooth muscle actin (α-SMA) has been established to be instrumental in the production of the contractile force during tissue contraction. The secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein involved in extracellular matrix (ECM) modification. The aim of this study is to investigate the molecular pathway involved in the induction of α-SMA expression when Tenons fibroblasts are cultured in stressed 3D collagen gels.
All experiments were performed on human Tenons fibroblasts. The cells were cultured in relaxed (control) and stressed 3D collagen I gels for 5 days. Knockdown of SPARC expression was performed by transfecting the cells with siRNA targeting SPARC (si-SPARC) and transfection with a scrambled siRNA was used as negative control. mRNA expressions for SPARC and α-SMA were determined by quantitative real-time PCR. Protein expressions of SPARC, α-SMA, Smad2 and phospho-Smad2 were detected by Western blotting.
α-SMA expression was increased in the stressed tenons fibroblasts relative to the relaxed ones. Corresponding increase in SPARC expression was also detected. However, when SPARC expression was knocked down with si-SPARC, there was no significant increase in α-SMA expression under stressed culture conditions. Additionally, the levels of phosphorylated Smad2 was found to be lower in the SPARC knocked-down cells compared to controls.
α-SMA is elevated under stressed conditions generated in the 3D collagen stress model. The presence of SPARC is necessary for the increased expression of α-SMA. We hypothesize that α-SMA expression under mechanically-induced stress requires signalling induced by SPARC and the TGFβ signalling pathway.
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