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.
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 1.5mg/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.


CONCLUSIONS
ETFB knockdown can affect TGF-β-induced tissue remodeling and/or fibrotic processes in vitro.
R&D Laboratories, POLA PHARMA INC., Yokohama, Japan. s-hirokawa@pola-pharma.co.jp
Shigenari
S
Hirokawa
Tomomasa
T
Shimanuki
Hiroyuki
H
Kitajima
Yasutomo
Y
Nishimori
Makoto
M
Shimosaka
D000199:Actins / Q000235:genetics*; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D017439:Cicatrix, Hypertrophic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D024042:Collagen Type I / Q000235:genetics*; D000090602:Collagen Type I, alpha 1 Chain; D015536:Down-Regulation; D044927:Electron-Transferring Flavoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D058628:Myofibroblasts / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D022502:Stress Fibers / Q000378:metabolism; D013314:Stress, Mechanical; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
C541116:ACTA2 protein, human; D000199:Actins; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; C500319:ETFB protein, human; D044927:Electron-Transferring Flavoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta