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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-β1induces the expression of the α-SMA and prolyl-4-hydroxylase. Human embryonic lung fibroblasts (HFL-1), incubated with or without TGF-β1 (10 ng/mL) for 24 h, were immunostained using antibodies against α-SMA (A) and prolyl-4-hydroxylase (C). Scale bars represent 50 μm. The α-SMA (B) and prolyl-4-hydroxylase (D) expression at 6, 24, and 48 h was examined by western blot and bands were quantified with densitometry. Presented values are the intensity of each band relative to loading control: GAPDH. Each value represent mean and SEM from four individual experiments. *P < 0.05.
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Figure 1: TGF-β1induces the expression of the α-SMA and prolyl-4-hydroxylase. Human embryonic lung fibroblasts (HFL-1), incubated with or without TGF-β1 (10 ng/mL) for 24 h, were immunostained using antibodies against α-SMA (A) and prolyl-4-hydroxylase (C). Scale bars represent 50 μm. The α-SMA (B) and prolyl-4-hydroxylase (D) expression at 6, 24, and 48 h was examined by western blot and bands were quantified with densitometry. Presented values are the intensity of each band relative to loading control: GAPDH. Each value represent mean and SEM from four individual experiments. *P < 0.05.

Mentions: TGF-β1 stimulation for 24 h increased the immuno-reactivity for α-SMA in human embryonic lung fibroblasts (HFL-1) cells, which was accompanied by a change in phenotype as shown by immunofluorescence (Figure 1A). These results were further confirmed by western blot that showed a significant two-fold increase of α-SMA upon TGF-β1 stimulation for 48 h (P < 0.05) (Figure 1B). Following TGF-β1 stimulation HFL-1 cells also had an increased immuno-reactivity for prolyl 4-hydroxylase (Figure 1C), which catalyzes post-translational formation of proline to 4-hydroxyproline in collagens. These results could not fully be confirmed by western blot, which showed a two-fold increase after 48 h (P < 0.06) (Figure 1D).


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-β1induces the expression of the α-SMA and prolyl-4-hydroxylase. Human embryonic lung fibroblasts (HFL-1), incubated with or without TGF-β1 (10 ng/mL) for 24 h, were immunostained using antibodies against α-SMA (A) and prolyl-4-hydroxylase (C). Scale bars represent 50 μm. The α-SMA (B) and prolyl-4-hydroxylase (D) expression at 6, 24, and 48 h was examined by western blot and bands were quantified with densitometry. Presented values are the intensity of each band relative to loading control: GAPDH. Each value represent mean and SEM from four individual experiments. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: TGF-β1induces the expression of the α-SMA and prolyl-4-hydroxylase. Human embryonic lung fibroblasts (HFL-1), incubated with or without TGF-β1 (10 ng/mL) for 24 h, were immunostained using antibodies against α-SMA (A) and prolyl-4-hydroxylase (C). Scale bars represent 50 μm. The α-SMA (B) and prolyl-4-hydroxylase (D) expression at 6, 24, and 48 h was examined by western blot and bands were quantified with densitometry. Presented values are the intensity of each band relative to loading control: GAPDH. Each value represent mean and SEM from four individual experiments. *P < 0.05.
Mentions: TGF-β1 stimulation for 24 h increased the immuno-reactivity for α-SMA in human embryonic lung fibroblasts (HFL-1) cells, which was accompanied by a change in phenotype as shown by immunofluorescence (Figure 1A). These results were further confirmed by western blot that showed a significant two-fold increase of α-SMA upon TGF-β1 stimulation for 48 h (P < 0.05) (Figure 1B). Following TGF-β1 stimulation HFL-1 cells also had an increased immuno-reactivity for prolyl 4-hydroxylase (Figure 1C), which catalyzes post-translational formation of proline to 4-hydroxyproline in collagens. These results could not fully be confirmed by western blot, which showed a two-fold increase after 48 h (P < 0.06) (Figure 1D).

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