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

BACKGROUND: The inhibition of transforming growth factor ß (TGF-ß)-induced myofibroblast differentiation is a key objective for the treatment of hypertrophic scarring. We previously reported that knockdown of the electron transfer flavoprotein ß 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-ß-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-ß 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-ß under mechanical stress conditions, ETFB knockdown attenuated α-SMA mRNA expression to a level comparable to that observed in the absence of TGF-ß. 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-ß. CONCLUSION: ETFB knockdown can affect TGF-ß-induced tissue remodeling and/or fibrotic processes in vitro.

Identification of ETFB as a candidate protein that participates in the mechanoregulation of fibroblast cell number in collagen gel culture.

BACKGROUND: Fibroblast activation is strongly influenced by mechanical environment in the wound-healing process, especially in fibrosis. Mechanically stressed three-dimensional collagen embedded culture is a useful model representing fibroblasts in morphological as well as biochemical situations encountered during fibrosis. OBJECTIVE: To find key proteins involved in reducing the number of fibroblasts during mechanical stress, we performed two-dimensional gel electrophoresis (2DE)-based differential display and siRNA-based functional screening with collagen gel culture focusing on the differences between attached and detached culture environments. METHODS: Membrane extracts of fibroblasts from 1 day of attached or detached cultures were subjected to 2DE. We compared protein expression levels and identified the attached-culture-dominant proteins by MALDI-TOF-MS. Next, fibroblasts were transfected with siRNA and embedded in collagen gel. Cell number was counted after 3 days in culture. RESULTS: Eight attached culture dominant proteins were identified with MALDI-TOF-MS. Transfection of siRNA against these proteins demonstrated that electron transfer flavoprotein ß subunit (ETFB)-specific siRNA reduced the cell number in the attached culture without a decrease in the detached culture. CONCLUSION: ETFB participates in the mechanoregulation of fibroblast cell number in collagen gel culture.

EBF-regulating Pax5 transcription is enhanced by STAT5 in the early stage of B cells.

Pax5 is an essential transcription factor for B cell development, and it is reported that Pax5 expression was reduced in the IL-7 receptor (IL-7R) knockout mouse. To investigate whether signals from the IL-7R regulate Pax5 transcription, we searched the consensus sequence of signal transducers and activators of transcription (STAT) in the Pax5 promoter region, since STAT is one of the components of cytokine signal transduction. A STAT-binding motif, termed SBM, was identified at 1,118 bp upstream of the transcriptional start site, and SBM completely overlapped with the binding site for early B cell factor (EBF). STAT5 was phosphorylated in the presence of IL-7 in the IL-7-dependent preB cell line, PreBR1, and phosphorylated-STAT5 as well as EBF was found to bind to the SBM. Moreover, we also revealed STAT5 binding to SBM in PreBR1 cells by chromatin immunoprecipitation assay. Transient co-transfection of reporter genes together with expression vectors of a constitutive active form of STAT5 and EBF into NIH3T3 cells demonstrated that STAT5 enhanced EBF-regulating transcription. Our results suggest that STAT5 phosphorylated by IL-7 can directly up-regulate Pax5 transcription in early B cells.