Knockdown of electron transfer flavoprotein β subunit reduced TGF-β-induced α-SMA mRNA expression but not COL1A1 in fibroblast-populated three-dimensional collagen gel cultures

摘要

Background: The inhibition of transforming growth factor β (TGF-β)-induced myofibroblast differentiation is a key objective for the treatment of hypertrophic scarring. We previously reported that knockdown of the electron transfer flavoprotein β subunit (ETFB) reduced mechanoregulated cell number in fibroblast-populated collagen gel cultures [1]. Objective: To characterize the effects of ETFB knockdown, we investigated gel contraction, TGF-β-induced collagen, α-SMA mRNA expression and stress fiber formation. Methods: Fibroblasts were transfected with negative control or ETFB-specific siRNAs and embedded in collagen gels in an attached or detached condition. The gel contraction assay was performed in three different concentrations of collagen (0.5, 1.0 or 1.5. mg/mL) and was analyzed by measuring the changes in the gel area throughout the culture period. The attached collagen gel culture was performed in the presence of rTGF-β and the mRNA levels of α-SMA and COL1A1 were measured by qRT-PCR. The effect of ETFB knockdown on proliferation and stress fiber organization in monolayer cultures was investigated by conducting AlamarBlue assays and phalloidin staining. Results: The transfection of ETFB siRNA did not alter gel contraction compared to the negative control in all collagen concentrations. When the cells were treated with TGF-β under mechanical stress conditions, ETFB knockdown attenuated α-SMA mRNA expression to a level comparable to that observed in the absence of TGF-β. However, no inhibitory effect on COL1A1 mRNA levels was observed. The AlamarBlue assay indicated that the knockdown had no effect on the proliferation of cells cultured on plastic. Phalloidin staining of a monolayer culture showed that ETFB knockdown weakened the stress fiber organization induced by rTGF-β. Conclusion: ETFB knockdown can affect TGF-β-induced tissue remodeling and/or fibrotic processes in vitro.