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Splicosomal and serine and arginine-rich splicing factors as targets for TGF-β.

Hallgren O, Malmström J, Malmström L, Andersson-Sjöland A, Wildt M, Tufvesson E, Juhasz P, Marko-Varga G, Westergren-Thorsson G - Fibrogenesis Tissue Repair (2012)

Bottom Line: Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection.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 results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden. oskar.hallgren@med.lu.se.

ABSTRACT

Background: Transforming 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 findings: The 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.

Conclusions: The 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.

No MeSH data available.


Related in: MedlinePlus

TGF-β1increases the relative expression of alternative splice forms of fibronectin. HFL-1 cells were incubated with or without TGF-β1 (10 ng/mL) for 6, 24, and 48 h and the relative expression of fibronectin-EDA compared to fibronectin were measured by q-PCR and western blot. (A) The relative expression of EDA fibronectin was compared to fibronectin by q-PCR. Each value represents mean and SEM from three individual experiments. (B) The relative expression of fibronectin-EDA compared to fibronectin was measured by western blot. Bands were quantified with densitometry and were related the loading control: GAPDH. Each value represents mean and SEM from four individual experiments. *P < 0.05. During the ECL development the 48-h fibronectin band became saturated. *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 2: TGF-β1increases the relative expression of alternative splice forms of fibronectin. HFL-1 cells were incubated with or without TGF-β1 (10 ng/mL) for 6, 24, and 48 h and the relative expression of fibronectin-EDA compared to fibronectin were measured by q-PCR and western blot. (A) The relative expression of EDA fibronectin was compared to fibronectin by q-PCR. Each value represents mean and SEM from three individual experiments. (B) The relative expression of fibronectin-EDA compared to fibronectin was measured by western blot. Bands were quantified with densitometry and were related the loading control: GAPDH. Each value represents mean and SEM from four individual experiments. *P < 0.05. During the ECL development the 48-h fibronectin band became saturated. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: TGF-β1 significantly increased the expression of both fibronectin and EDA fibronectin. However, following TGF-β1 stimulation the relative ratio between EDA fibronectin and fibronectin was significantly increased over time as shown by q-PCR (Figure 2A). 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). These data show that TGF-β1 may induce myofibroblast differentiation and this is accompanied by an altered splicing pattern, exemplified by the increase in alternatively spliced isoforms of fibronectin.


Splicosomal and serine and arginine-rich splicing factors as targets for TGF-β.

Hallgren O, Malmström J, Malmström L, Andersson-Sjöland A, Wildt M, Tufvesson E, Juhasz P, Marko-Varga G, Westergren-Thorsson G - Fibrogenesis Tissue Repair (2012)

TGF-β1increases the relative expression of alternative splice forms of fibronectin. HFL-1 cells were incubated with or without TGF-β1 (10 ng/mL) for 6, 24, and 48 h and the relative expression of fibronectin-EDA compared to fibronectin were measured by q-PCR and western blot. (A) The relative expression of EDA fibronectin was compared to fibronectin by q-PCR. Each value represents mean and SEM from three individual experiments. (B) The relative expression of fibronectin-EDA compared to fibronectin was measured by western blot. Bands were quantified with densitometry and were related the loading control: GAPDH. Each value represents mean and SEM from four individual experiments. *P < 0.05. During the ECL development the 48-h fibronectin band became saturated. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472233&req=5

Figure 2: TGF-β1increases the relative expression of alternative splice forms of fibronectin. HFL-1 cells were incubated with or without TGF-β1 (10 ng/mL) for 6, 24, and 48 h and the relative expression of fibronectin-EDA compared to fibronectin were measured by q-PCR and western blot. (A) The relative expression of EDA fibronectin was compared to fibronectin by q-PCR. Each value represents mean and SEM from three individual experiments. (B) The relative expression of fibronectin-EDA compared to fibronectin was measured by western blot. Bands were quantified with densitometry and were related the loading control: GAPDH. Each value represents mean and SEM from four individual experiments. *P < 0.05. During the ECL development the 48-h fibronectin band became saturated. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: TGF-β1 significantly increased the expression of both fibronectin and EDA fibronectin. However, following TGF-β1 stimulation the relative ratio between EDA fibronectin and fibronectin was significantly increased over time as shown by q-PCR (Figure 2A). 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). These data show that TGF-β1 may induce myofibroblast differentiation and this is accompanied by an altered splicing pattern, exemplified by the increase in alternatively spliced isoforms of fibronectin.

Bottom Line: Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection.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 results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden. oskar.hallgren@med.lu.se.

ABSTRACT

Background: Transforming 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 findings: The 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.

Conclusions: The 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.

No MeSH data available.


Related in: MedlinePlus