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Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Manabe R, Ohe N, Maeda T, Fukuda T, Sekiguchi K - J. Cell Biol. (1997)

Bottom Line: To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins.Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module.Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, Osaka Medical Center for Maternal and Child Health, Japan.

ABSTRACT
Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

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Migration of HT1080 cells on recombinant FNs. 96-well plates were precoated with 5 μg/ml of rFN(C) (open bars),  rFN(AC) (closed bars), or rFN(BAC) (hatched bars) and then  partially covered with plastic discs (φ 6 mm) that had been cut in  half and coated with 0.01% poly-l-lysine. HT1080 cells were  seeded onto the plates and incubated for 1.5 h at 37°C to allow  them to spread. The plastic discs were then removed and the cells  were further incubated at 37°C for 12 h to allow them to migrate  into the open space left after removal of the discs. (A) The cells  were photographed before and after migration at 37°C for 12 h.  The positions of the cell front before and after the cell migration  were indicated by open and closed arrowheads, respectively. (B)  Cell motility on different substrates was quantified by measuring  the distance of outward migration, i.e., the distance between the  positions of the cell front before and after cell migration. Cell  motility was assayed in the presence or absence of 20 μg/ml of the  anti-integrin α5 subunit mAb 8F1 or control IgG. Each bar represents the mean ± SD (n = 6).
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Figure 11: Migration of HT1080 cells on recombinant FNs. 96-well plates were precoated with 5 μg/ml of rFN(C) (open bars), rFN(AC) (closed bars), or rFN(BAC) (hatched bars) and then partially covered with plastic discs (φ 6 mm) that had been cut in half and coated with 0.01% poly-l-lysine. HT1080 cells were seeded onto the plates and incubated for 1.5 h at 37°C to allow them to spread. The plastic discs were then removed and the cells were further incubated at 37°C for 12 h to allow them to migrate into the open space left after removal of the discs. (A) The cells were photographed before and after migration at 37°C for 12 h. The positions of the cell front before and after the cell migration were indicated by open and closed arrowheads, respectively. (B) Cell motility on different substrates was quantified by measuring the distance of outward migration, i.e., the distance between the positions of the cell front before and after cell migration. Cell motility was assayed in the presence or absence of 20 μg/ml of the anti-integrin α5 subunit mAb 8F1 or control IgG. Each bar represents the mean ± SD (n = 6).

Mentions: FN is known to promote cell migration via interaction with integrin α5β1 (Yamada et al., 1990). Increased binding avidity of integrin α5β1 for EDA+ FN may, therefore, lead to an enhanced cell motility on substratum coated with EDA+ FN. To test this possibility, migration of HT1080 cells on the substrates coated with EDA+ or EDA− FNs were compared (Fig. 11 A). HT1080 cells were significantly more migratory on rFN(AC) and rFN(BAC) than on rFN(C). No significant cell migration was observed on substrates coated with poly-l-lysine (data not shown). Quantitation of outward cell migration showed that HT1080 cells migrated 1.7–2 times farther on substrates coated with rFN(AC) or rFN(BAC) than on the substrate coated with rFN(C) (Fig. 11 B). Cell migration mediated by EDA+ and EDA− FNs was inhibited by anti-integrin α5 mAb 8F1 to a similar extent, suggesting that increased cell motility on the EDA+ FNs was due to the increased integrin recognition of CCBD flanked with the EDA segment.


Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Manabe R, Ohe N, Maeda T, Fukuda T, Sekiguchi K - J. Cell Biol. (1997)

Migration of HT1080 cells on recombinant FNs. 96-well plates were precoated with 5 μg/ml of rFN(C) (open bars),  rFN(AC) (closed bars), or rFN(BAC) (hatched bars) and then  partially covered with plastic discs (φ 6 mm) that had been cut in  half and coated with 0.01% poly-l-lysine. HT1080 cells were  seeded onto the plates and incubated for 1.5 h at 37°C to allow  them to spread. The plastic discs were then removed and the cells  were further incubated at 37°C for 12 h to allow them to migrate  into the open space left after removal of the discs. (A) The cells  were photographed before and after migration at 37°C for 12 h.  The positions of the cell front before and after the cell migration  were indicated by open and closed arrowheads, respectively. (B)  Cell motility on different substrates was quantified by measuring  the distance of outward migration, i.e., the distance between the  positions of the cell front before and after cell migration. Cell  motility was assayed in the presence or absence of 20 μg/ml of the  anti-integrin α5 subunit mAb 8F1 or control IgG. Each bar represents the mean ± SD (n = 6).
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Figure 11: Migration of HT1080 cells on recombinant FNs. 96-well plates were precoated with 5 μg/ml of rFN(C) (open bars), rFN(AC) (closed bars), or rFN(BAC) (hatched bars) and then partially covered with plastic discs (φ 6 mm) that had been cut in half and coated with 0.01% poly-l-lysine. HT1080 cells were seeded onto the plates and incubated for 1.5 h at 37°C to allow them to spread. The plastic discs were then removed and the cells were further incubated at 37°C for 12 h to allow them to migrate into the open space left after removal of the discs. (A) The cells were photographed before and after migration at 37°C for 12 h. The positions of the cell front before and after the cell migration were indicated by open and closed arrowheads, respectively. (B) Cell motility on different substrates was quantified by measuring the distance of outward migration, i.e., the distance between the positions of the cell front before and after cell migration. Cell motility was assayed in the presence or absence of 20 μg/ml of the anti-integrin α5 subunit mAb 8F1 or control IgG. Each bar represents the mean ± SD (n = 6).
Mentions: FN is known to promote cell migration via interaction with integrin α5β1 (Yamada et al., 1990). Increased binding avidity of integrin α5β1 for EDA+ FN may, therefore, lead to an enhanced cell motility on substratum coated with EDA+ FN. To test this possibility, migration of HT1080 cells on the substrates coated with EDA+ or EDA− FNs were compared (Fig. 11 A). HT1080 cells were significantly more migratory on rFN(AC) and rFN(BAC) than on rFN(C). No significant cell migration was observed on substrates coated with poly-l-lysine (data not shown). Quantitation of outward cell migration showed that HT1080 cells migrated 1.7–2 times farther on substrates coated with rFN(AC) or rFN(BAC) than on the substrate coated with rFN(C) (Fig. 11 B). Cell migration mediated by EDA+ and EDA− FNs was inhibited by anti-integrin α5 mAb 8F1 to a similar extent, suggesting that increased cell motility on the EDA+ FNs was due to the increased integrin recognition of CCBD flanked with the EDA segment.

Bottom Line: To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins.Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module.Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, Osaka Medical Center for Maternal and Child Health, Japan.

ABSTRACT
Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

Show MeSH
Related in: MedlinePlus