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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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Attachment and  spreading of HT1080 cells on  FN-coated substratum. (A)  HT1080 cells (2 × 104) were  seeded on 96-well microtiter  plates coated with 5 μg/ml of  different FN isoforms and incubated for 30 min at 37°C.  The cells were rinsed, fixed,  and stained with Giemsa.  Bar, 100 μm. (B) Spreading  of HT1080 cells was quantified as described in Materials  and Methods. The standard  deviations of multiple determinations (n = 3) are indicated at the top of bars.
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Figure 3: Attachment and spreading of HT1080 cells on FN-coated substratum. (A) HT1080 cells (2 × 104) were seeded on 96-well microtiter plates coated with 5 μg/ml of different FN isoforms and incubated for 30 min at 37°C. The cells were rinsed, fixed, and stained with Giemsa. Bar, 100 μm. (B) Spreading of HT1080 cells was quantified as described in Materials and Methods. The standard deviations of multiple determinations (n = 3) are indicated at the top of bars.

Mentions: EDA+ FN isoforms have been shown to be expressed prominently in tissues where cells actively proliferate and migrate, such as those in embryos, tumors, and healing wounds. To explore the physiological functions of the EDA segment, we first compared the cell-adhesive activity of recombinant FNs with or without EDA. When HT1080 cells were incubated on substrates coated with recombinant FNs or with plasma or cellular FN, significant differences were seen in the numbers of cells attached to different forms of FNs (Fig. 3 A). HT1080 cells attached in greater numbers to substrates coated with rFN(AC) or rFN(BAC) than to the substrate coated with rFN(C). A similar but less pronounced difference was observed between the substrates coated with plasma or cellular FNs. In addition to promoting more cell attachment, rFN(AC) and rFN(BAC) were more potent in inducing cell spreading than rFN(C) (Fig. 3 B). Similarly, cellular FN was more active than plasma FN in inducing cell spreading, although the difference between cellular and plasma FNs was less evident than between rFN(AC) and rFN(C). No significant difference was found, however, between rFN(AC) and rFN(BAC), indicating that the insertion of the EDB segment did not affect the cell spreading activity of EDA+ FN isoforms. Enhanced cell spreading onto EDA+ FN-coated substrates was also observed with other cell lines including rat NRK cells, mouse L cells, and hamster CHO-K1 cells (data not shown).


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)

Attachment and  spreading of HT1080 cells on  FN-coated substratum. (A)  HT1080 cells (2 × 104) were  seeded on 96-well microtiter  plates coated with 5 μg/ml of  different FN isoforms and incubated for 30 min at 37°C.  The cells were rinsed, fixed,  and stained with Giemsa.  Bar, 100 μm. (B) Spreading  of HT1080 cells was quantified as described in Materials  and Methods. The standard  deviations of multiple determinations (n = 3) are indicated at the top of bars.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139828&req=5

Figure 3: Attachment and spreading of HT1080 cells on FN-coated substratum. (A) HT1080 cells (2 × 104) were seeded on 96-well microtiter plates coated with 5 μg/ml of different FN isoforms and incubated for 30 min at 37°C. The cells were rinsed, fixed, and stained with Giemsa. Bar, 100 μm. (B) Spreading of HT1080 cells was quantified as described in Materials and Methods. The standard deviations of multiple determinations (n = 3) are indicated at the top of bars.
Mentions: EDA+ FN isoforms have been shown to be expressed prominently in tissues where cells actively proliferate and migrate, such as those in embryos, tumors, and healing wounds. To explore the physiological functions of the EDA segment, we first compared the cell-adhesive activity of recombinant FNs with or without EDA. When HT1080 cells were incubated on substrates coated with recombinant FNs or with plasma or cellular FN, significant differences were seen in the numbers of cells attached to different forms of FNs (Fig. 3 A). HT1080 cells attached in greater numbers to substrates coated with rFN(AC) or rFN(BAC) than to the substrate coated with rFN(C). A similar but less pronounced difference was observed between the substrates coated with plasma or cellular FNs. In addition to promoting more cell attachment, rFN(AC) and rFN(BAC) were more potent in inducing cell spreading than rFN(C) (Fig. 3 B). Similarly, cellular FN was more active than plasma FN in inducing cell spreading, although the difference between cellular and plasma FNs was less evident than between rFN(AC) and rFN(C). No significant difference was found, however, between rFN(AC) and rFN(BAC), indicating that the insertion of the EDB segment did not affect the cell spreading activity of EDA+ FN isoforms. Enhanced cell spreading onto EDA+ FN-coated substrates was also observed with other cell lines including rat NRK cells, mouse L cells, and hamster CHO-K1 cells (data not shown).

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