Biochemical and Biophysical Research Communications
Smad3 mediates TGF-β1-induced collagen gel contraction by human lung fibroblasts
Section snippets
Materials and methods
Cell culture. Human fetal lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in 100-mm tissue culture dishes (Falcon; Becton–Dickinson Labware, Lincoln Park, NJ) in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (FCS), 50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate (penicillin–streptomycin; Invitrogen, Life Technologies, Grand Island, NY), and 1 μg/ml amphotericin B (pharma-Tek,
Smad3 mediates the TGF-β1-induced collagen gel contraction by human lung fibroblasts
Inhibition of HFL-1 Smad2 and Smad3 protein expression after treating with the siRNA used has been assessed by Western blot and previously reported [7]. To evaluate transcriptional activation of Smad3, we used reporter constructs specific for Smad3 (CAGA12) [10], [11]. As expected, transfection with Smad3-siRNA resulted in almost 100% reduction in TGF-β1-stimulated CAGA12 reporter activity. This extends the previous results obtained by Western blot and demonstrates inhibition of TGF-β-induced
Discussion
Fibroblasts cultured in a three-dimensional collagen matrix attach to the collagen fibers and generate mechanical tension [5], [6]. If maintained in floating culture, fibroblasts will cause the surrounding matrix to contract [12]. This in vitro effect is believed to be a model of the contraction that characterizes both wound healing and the development of fibrosis. Transforming growth factor beta (TGF-β) stimulates the process [13]. The current study demonstrates that, in human fibroblasts, the
Acknowledgments
We appreciate the helpful discussions with Dr. A. Roberts, National Institutes of Health (NIH), and the secretarial support of Ms. Lillian Richards. This work was supported by NIH Grant 5 R01 HL64088-04.
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