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Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE - J. Cell Biol. (2003)

Bottom Line: However, the precise role of the FN isoforms is poorly understood.One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging.Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.

ABSTRACT
Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

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The decrease in FN levels in tissues of EDA+/+ mice is not correlated to a reduction in integrin levels. (A and B) Western blot analysis of total protein extracts (50 μg) from brain, heart, lung, and liver from all genotypes (EDAwt/wt, EDA+/+, EDA−/−, EDA+/−, EDA−/wt, and EDA+/wt) using anti-FN polyclonal antibody (A) and, after stripping the membrane, anti–β-tubulin mAb to verify for minor errors in protein load (B). Note that the migration of the EDA+ FN is slightly slower than that of the EDA− FN. Coomassie blue staining of 5–17% gradient gels of the same protein extracts did not show changes in other proteins. (C) Plasma and serum protein samples from EDA+/+, EDA−/−, and EDAwt/wt mice (20 μg) were Coomassie blue stained (left), and a similar gel was blotted and incubated with anti-FN antibody (middle). For the anti-EDA antibody (right), 100 μg of protein extract were loaded. (D and E) Western blot analysis of α9- and β1-integrin levels in different tissues (liver, brain, and skin) using anti-α9 and -β1 rabbit polyclonal antibodies. Anti–β-tubulin mAb was used to verify for minor errors in protein load.
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fig3: The decrease in FN levels in tissues of EDA+/+ mice is not correlated to a reduction in integrin levels. (A and B) Western blot analysis of total protein extracts (50 μg) from brain, heart, lung, and liver from all genotypes (EDAwt/wt, EDA+/+, EDA−/−, EDA+/−, EDA−/wt, and EDA+/wt) using anti-FN polyclonal antibody (A) and, after stripping the membrane, anti–β-tubulin mAb to verify for minor errors in protein load (B). Note that the migration of the EDA+ FN is slightly slower than that of the EDA− FN. Coomassie blue staining of 5–17% gradient gels of the same protein extracts did not show changes in other proteins. (C) Plasma and serum protein samples from EDA+/+, EDA−/−, and EDAwt/wt mice (20 μg) were Coomassie blue stained (left), and a similar gel was blotted and incubated with anti-FN antibody (middle). For the anti-EDA antibody (right), 100 μg of protein extract were loaded. (D and E) Western blot analysis of α9- and β1-integrin levels in different tissues (liver, brain, and skin) using anti-α9 and -β1 rabbit polyclonal antibodies. Anti–β-tubulin mAb was used to verify for minor errors in protein load.

Mentions: Next, we examined whether the modifications in the natural alternative splicing pattern of FN had some consequences in other cellular proteins or FN itself. Consequently, protein extracts from different tissues of the different genotypes were prepared from 3-mo-old mice and analyzed by SDS-PAGE. No evident difference was observed at least at the sensitivity level of Coomassie blue staining of 5–17% gradient gels (unpublished data). On the other hand, a Western blot analysis using anti-FN polyclonal antibody showed a striking decrease in FN levels in most of the tissues analyzed from EDA+/+ mice (Fig. 3Figure 3.


Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE - J. Cell Biol. (2003)

The decrease in FN levels in tissues of EDA+/+ mice is not correlated to a reduction in integrin levels. (A and B) Western blot analysis of total protein extracts (50 μg) from brain, heart, lung, and liver from all genotypes (EDAwt/wt, EDA+/+, EDA−/−, EDA+/−, EDA−/wt, and EDA+/wt) using anti-FN polyclonal antibody (A) and, after stripping the membrane, anti–β-tubulin mAb to verify for minor errors in protein load (B). Note that the migration of the EDA+ FN is slightly slower than that of the EDA− FN. Coomassie blue staining of 5–17% gradient gels of the same protein extracts did not show changes in other proteins. (C) Plasma and serum protein samples from EDA+/+, EDA−/−, and EDAwt/wt mice (20 μg) were Coomassie blue stained (left), and a similar gel was blotted and incubated with anti-FN antibody (middle). For the anti-EDA antibody (right), 100 μg of protein extract were loaded. (D and E) Western blot analysis of α9- and β1-integrin levels in different tissues (liver, brain, and skin) using anti-α9 and -β1 rabbit polyclonal antibodies. Anti–β-tubulin mAb was used to verify for minor errors in protein load.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172721&req=5

fig3: The decrease in FN levels in tissues of EDA+/+ mice is not correlated to a reduction in integrin levels. (A and B) Western blot analysis of total protein extracts (50 μg) from brain, heart, lung, and liver from all genotypes (EDAwt/wt, EDA+/+, EDA−/−, EDA+/−, EDA−/wt, and EDA+/wt) using anti-FN polyclonal antibody (A) and, after stripping the membrane, anti–β-tubulin mAb to verify for minor errors in protein load (B). Note that the migration of the EDA+ FN is slightly slower than that of the EDA− FN. Coomassie blue staining of 5–17% gradient gels of the same protein extracts did not show changes in other proteins. (C) Plasma and serum protein samples from EDA+/+, EDA−/−, and EDAwt/wt mice (20 μg) were Coomassie blue stained (left), and a similar gel was blotted and incubated with anti-FN antibody (middle). For the anti-EDA antibody (right), 100 μg of protein extract were loaded. (D and E) Western blot analysis of α9- and β1-integrin levels in different tissues (liver, brain, and skin) using anti-α9 and -β1 rabbit polyclonal antibodies. Anti–β-tubulin mAb was used to verify for minor errors in protein load.
Mentions: Next, we examined whether the modifications in the natural alternative splicing pattern of FN had some consequences in other cellular proteins or FN itself. Consequently, protein extracts from different tissues of the different genotypes were prepared from 3-mo-old mice and analyzed by SDS-PAGE. No evident difference was observed at least at the sensitivity level of Coomassie blue staining of 5–17% gradient gels (unpublished data). On the other hand, a Western blot analysis using anti-FN polyclonal antibody showed a striking decrease in FN levels in most of the tissues analyzed from EDA+/+ mice (Fig. 3Figure 3.

Bottom Line: However, the precise role of the FN isoforms is poorly understood.One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging.Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.

ABSTRACT
Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

Show MeSH
Related in: MedlinePlus