Downregulation of activin‑signaling gene expression in passaged normal human dermal fibroblasts

Abstract

Activins are members of the transforming growth factor-β (TGF-β) superfamily and play important roles in proliferation, differentiation, and apoptosis of various target cells. We investigated changes of activin, activin receptor (ActR), and Smad-signaling gene expression with increasing passage number in normal human dermal fibroblasts. The expression of mRNA and protein was measured by reverse transcription-quantitative polymerase chain reaction and immunoblot analysis from passage numbers 5 to 15. Activin A and follistatin transcript levels increased with increasing passage number. ActR types IA, IB, IIA and IIB mRNA levels decreased at high passage number. The levels of Smad2, 3 and 4 protein decreased with increasing passage number, which also attenuated phosphorylation of Smad2 and 3 protein expression. Smad7 was enhanced with increasing passage number. These results suggest that expression of activin-signaling in aging normal human dermal fibroblasts increases activin A and follistatin, whereas ActR-Smad signaling is decreased.

Bioactive Fraction of Aronia melanocarpa Fruit Inhibits Adipogenic Differentiation of Cultured 3T3-L1 Cells

“…The relationship between the target gene and GAPDH, serving as a housekeeping gene, was determined using the formula 2 -(target gene-GAPDH) . The relative quantities of transcripts were measured [25].…”

“…Cells were harvested, washed with cold PBS, and treated with lysis buffer containing 1 mM PMSF (Cell Signaling Technology, Inc.). According to the manufacturer's protocol, protein concentrations were determined using a BCA protein assay (Thermo Fisher Scientific, Inc.) [25]. The protein extracts (10 µg per lane) were fractionated on 10% or 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Inc., Buckinghamshire, UK).…”