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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

Cellular localization of SR proteins. HFL-1 cells were incubated for 24 and 48 h with or without TGF-β1 (10 ng/mL). The intracellular localization of SR proteins was examined with immunofluorescence using an antibody that recognizes a phospho-epitope on SR proteins. Staining for SR proteins are shown in red and nucleus are visualized by DAPI (blue). Immune-reactivity for SR proteins was exclusively observed in cell nucleus. Arrowheads show speckles with more intense staining.
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Figure 7: Cellular localization of SR proteins. HFL-1 cells were incubated for 24 and 48 h with or without TGF-β1 (10 ng/mL). The intracellular localization of SR proteins was examined with immunofluorescence using an antibody that recognizes a phospho-epitope on SR proteins. Staining for SR proteins are shown in red and nucleus are visualized by DAPI (blue). Immune-reactivity for SR proteins was exclusively observed in cell nucleus. Arrowheads show speckles with more intense staining.

Mentions: Next, we investigated how TGF-β1 stimulation influenced the cellular localization of SR proteins and SRp20 by immunofluorescence. The staining pattern of the splicing factors, using the anti-phospho SR proteins antibody, was exclusively nuclear (Figure 7). The staining was more intense at 48 h but this could surprisingly not be observed at 24 h of TGF-β1 stimulation compared to untreated cells. In addition the staining at 48 h was more speckled but not to the same extent at 24 h TGF-β1 stimulation. The staining when using the SRp20 antibody was mainly nuclear in untreated cells and after 24 h of TGF-β1 stimulation although some faint extra-nuclear staining also could be observed ( 8). However, after 48 h TGF-β1 stimulation SRp20 was both located intra- and extra-nuclear and in addition the staining was more speckled and intense.


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)

Cellular localization of SR proteins. HFL-1 cells were incubated for 24 and 48 h with or without TGF-β1 (10 ng/mL). The intracellular localization of SR proteins was examined with immunofluorescence using an antibody that recognizes a phospho-epitope on SR proteins. Staining for SR proteins are shown in red and nucleus are visualized by DAPI (blue). Immune-reactivity for SR proteins was exclusively observed in cell nucleus. Arrowheads show speckles with more intense staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Cellular localization of SR proteins. HFL-1 cells were incubated for 24 and 48 h with or without TGF-β1 (10 ng/mL). The intracellular localization of SR proteins was examined with immunofluorescence using an antibody that recognizes a phospho-epitope on SR proteins. Staining for SR proteins are shown in red and nucleus are visualized by DAPI (blue). Immune-reactivity for SR proteins was exclusively observed in cell nucleus. Arrowheads show speckles with more intense staining.
Mentions: Next, we investigated how TGF-β1 stimulation influenced the cellular localization of SR proteins and SRp20 by immunofluorescence. The staining pattern of the splicing factors, using the anti-phospho SR proteins antibody, was exclusively nuclear (Figure 7). The staining was more intense at 48 h but this could surprisingly not be observed at 24 h of TGF-β1 stimulation compared to untreated cells. In addition the staining at 48 h was more speckled but not to the same extent at 24 h TGF-β1 stimulation. The staining when using the SRp20 antibody was mainly nuclear in untreated cells and after 24 h of TGF-β1 stimulation although some faint extra-nuclear staining also could be observed ( 8). However, after 48 h TGF-β1 stimulation SRp20 was both located intra- and extra-nuclear and in addition the staining was more speckled and intense.

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