Abstract
BackgroundTransforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins.Methodology/Principal findingsThe expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/− 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence.ConclusionsThe results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.
Highlights
Transforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation
TGF-β1 induces myofibroblast-like characteristics in fibroblasts TGF-β1 stimulation for 24 h increased the immuno-reactivity for α-smooth muscle actin (α-SMA) in human embryonic lung fibroblasts (HFL-1) cells, which was accompanied by a change in phenotype as shown by immunofluorescence (Figure 1A)
This was confirmed by western blot showing that TGF-β1 increased the relative expression of EDA fibronectin compared to fibronectin from 269% at 24 h to (P < 0.01) to 526% at 48 h (P < 0.001) (Figure 2B)
Summary
Transforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. The growth factor TGF-β1 has been shown to play an important role in the differentiation process inducing the During constitutive and alternative splicing of gene products, splice site selection is regulated by altering initial binding of serine-arginine-rich splicing factors (SR proteins) to pre-mRNA. These factors contain an N-terminal RNA recognition motif that allows binding to pre-mRNA and a C-terminal serine-arginine-rich domain that mediates protein-protein interactions. Hypo-phosphorylation of one domain of SR proteins serves as a nuclear export signal [22]
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