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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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Effects of anti-EDA mAbs and recombinant EDA  fragments on cell spreading mediated by recombinant FNs. (A)  Microtiter plates precoated with 5 μg/ml of rFN(C) (open bars)  or rFN(AC) (closed bars) were treated with the following mAbs  (20 μg/ml) at 37°C for 30 min before the addition of HT1080 cells:  None, no addition of mAbs; IST-9 and HHS-01, two different  anti-EDA mAbs; FN12-8+FN30-8, a mixture of two function-blocking mAbs directed to CCBD. The cells were incubated at  37°C for 30 min and the number of cells adopting a well spread  morphology was determined. (B) In separate experiments,  HT1080 cells were seeded onto microtiter plates precoated with 5  μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) and incubated for 30 min at 37°C in the presence or absence of MBP fusion fragments with (MBP11-A-12) and without (MBP-11-12) the  EDA segment. Each bar represents the mean ± SD (n = 6).
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Figure 5: Effects of anti-EDA mAbs and recombinant EDA fragments on cell spreading mediated by recombinant FNs. (A) Microtiter plates precoated with 5 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) were treated with the following mAbs (20 μg/ml) at 37°C for 30 min before the addition of HT1080 cells: None, no addition of mAbs; IST-9 and HHS-01, two different anti-EDA mAbs; FN12-8+FN30-8, a mixture of two function-blocking mAbs directed to CCBD. The cells were incubated at 37°C for 30 min and the number of cells adopting a well spread morphology was determined. (B) In separate experiments, HT1080 cells were seeded onto microtiter plates precoated with 5 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) and incubated for 30 min at 37°C in the presence or absence of MBP fusion fragments with (MBP11-A-12) and without (MBP-11-12) the EDA segment. Each bar represents the mean ± SD (n = 6).

Mentions: One possibility to explain enhanced cell speading on the EDA+ FN isoforms is that the EDA segment may contain an additional cell-interactive site that cooperates additively or synergistically with the RGD motif in CCBD. To explore this possibility, two types of EDA antagonists, i.e., mAbs directed against the EDA segment and recombinant peptide containing the EDA segment, were tested for their abilities to inhibit rFN(AC)-mediated cell spreading. Pretreatment with two distinct anti-EDA mAbs (IST-9 and HHS01) did not inhibit cell spreading on rFN(AC) or rFN(C), whereas the function-blocking mAbs directed against CCBD inhibited cell spreading almost completely on both types of FN isoforms (Fig. 5 A). Furthermore, recombinant peptide MBP11-A-12 containing the EDA segment failed to inhibit cell spreading onto the rFN(AC)- coated substrates, as was the case with the control MBP fusion protein lacking the EDA segment (Fig. 5 B). The inability of the EDA segment to directly interact with HT1080 cells was also supported by the observation that neither MBP11-A-12 nor MBP11-12 could mediate adhesion of HT1080 cells (data not shown). These results, taken together, indicate that the EDA segment is unlikely to be directly involved in enhanced cell adhesion onto the EDA+ FN-coated substrates as an independent cell-interactive site.


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)

Effects of anti-EDA mAbs and recombinant EDA  fragments on cell spreading mediated by recombinant FNs. (A)  Microtiter plates precoated with 5 μg/ml of rFN(C) (open bars)  or rFN(AC) (closed bars) were treated with the following mAbs  (20 μg/ml) at 37°C for 30 min before the addition of HT1080 cells:  None, no addition of mAbs; IST-9 and HHS-01, two different  anti-EDA mAbs; FN12-8+FN30-8, a mixture of two function-blocking mAbs directed to CCBD. The cells were incubated at  37°C for 30 min and the number of cells adopting a well spread  morphology was determined. (B) In separate experiments,  HT1080 cells were seeded onto microtiter plates precoated with 5  μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) and incubated for 30 min at 37°C in the presence or absence of MBP fusion fragments with (MBP11-A-12) and without (MBP-11-12) the  EDA segment. Each bar represents the mean ± SD (n = 6).
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Figure 5: Effects of anti-EDA mAbs and recombinant EDA fragments on cell spreading mediated by recombinant FNs. (A) Microtiter plates precoated with 5 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) were treated with the following mAbs (20 μg/ml) at 37°C for 30 min before the addition of HT1080 cells: None, no addition of mAbs; IST-9 and HHS-01, two different anti-EDA mAbs; FN12-8+FN30-8, a mixture of two function-blocking mAbs directed to CCBD. The cells were incubated at 37°C for 30 min and the number of cells adopting a well spread morphology was determined. (B) In separate experiments, HT1080 cells were seeded onto microtiter plates precoated with 5 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) and incubated for 30 min at 37°C in the presence or absence of MBP fusion fragments with (MBP11-A-12) and without (MBP-11-12) the EDA segment. Each bar represents the mean ± SD (n = 6).
Mentions: One possibility to explain enhanced cell speading on the EDA+ FN isoforms is that the EDA segment may contain an additional cell-interactive site that cooperates additively or synergistically with the RGD motif in CCBD. To explore this possibility, two types of EDA antagonists, i.e., mAbs directed against the EDA segment and recombinant peptide containing the EDA segment, were tested for their abilities to inhibit rFN(AC)-mediated cell spreading. Pretreatment with two distinct anti-EDA mAbs (IST-9 and HHS01) did not inhibit cell spreading on rFN(AC) or rFN(C), whereas the function-blocking mAbs directed against CCBD inhibited cell spreading almost completely on both types of FN isoforms (Fig. 5 A). Furthermore, recombinant peptide MBP11-A-12 containing the EDA segment failed to inhibit cell spreading onto the rFN(AC)- coated substrates, as was the case with the control MBP fusion protein lacking the EDA segment (Fig. 5 B). The inability of the EDA segment to directly interact with HT1080 cells was also supported by the observation that neither MBP11-A-12 nor MBP11-12 could mediate adhesion of HT1080 cells (data not shown). These results, taken together, indicate that the EDA segment is unlikely to be directly involved in enhanced cell adhesion onto the EDA+ FN-coated substrates as an independent cell-interactive site.

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