Functional Characterization of Transforming Growth Factor β Signaling in Smad2- and Smad3-deficient Fibroblasts

Abstract

A prominent pathway of transforming growth factor (TGF)-␤ signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-␤1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-␤1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-␤1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [ 3 H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-␤1. Lack of Smad2 or Smad3 expression did not affect TGF-␤1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-␤1. Moreover, TGF-␤1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-␤1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-␤-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element) 4 -Lux reporter by TGF-␤1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-␤1 signaling.

The role of TGF-β and epithelial-to mesenchymal transition in diabetic nephropathy

“…For example, TGF-β1 mRNA auto-induction is thought to have a beneficial role in cardiac wound healing after ischemic injury [44] , whilst auto-induction of TGF-β1 at the site of injury in the proximal tubule may result in a positive feedback, where sustained cytokine production may accelerate EMT and increase the generation of myofibroblasts thus promoting and exacerbating fibrosis. A correlation between Smads and AP-1 has also been suggested following elegant studies performed in a mouse model of unilateral ureteric obstruction and in fibroblasts obtained from Smad2 and Smad3 knockout animals [45][46]. These studies confirmed not only a correlation between Smad signalling and TGF-β1 auto-induction, but further demonstrated that the relationship depended on the presence and activation of Smad3.…”

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

“…In normal fibroblasts, SMADs function as essential cytoplasmic effectors for TGF␤ stimulation of collagen synthesis, as well as that of CTGF (40) and of TGF␤ itself (41). Genetic targeting of the SMAD3 locus protected null mice from the development of radiationinduced fibrosis, indicating the fundamental physiologic role of SMAD3 in processes of tissue repair and fibrosis (42).…”

Smad3 deficiency inhibits v-ras-induced transformation by suppression of JNK MAPK signaling and increased farnesyl transferase inhibition

“…A dramatically reduced level of phospho-JNK was seen in TGF-b-treated S3KO cells compared to S3WT MEFs (Figure 1b Immunoblotting was performed with 20-50 mg of samples using Tris-Glycine gels (Invitrogen, Carlsbad, CA, USA), 0.2 mm Nitrocellulose (Amersham, Piscataway, NJ, USA) and incubated with primary antibodies against Phospho-JNK, Phospho-ERK, total JNK and total ERK (all Cell Signaling, Danvers, MA, USA) followed by anti-rabbit HRP secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and chemiluminescence (ECL Plus, Pierce Biotechnology Inc., Rockford, IL, USA) was detected by films (Kodak MR, Sigma-Aldrich, St Louis, MO, USA) or digital imager (AlphaInotech, San Leandro, CA, USA). Primary mouse embryonic fibroblasts (b-d) were isolated from wild-type (S3WT), Heterozygote (S3Het) and knockout (S3KO) embryos between 12 and 16 days of gestation (Piek et al, 2001), propagated in Dulbecco's-modified Eagle's medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA), grown to confluence and used between 2 and7 passages for all assays. Blots are representative of atleast three independent experiments.…”