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Growth factor-dependent activation of alphavbeta3 integrin in normal epithelial cells: implications for tumor invasion.

Trusolino L, Serini G, Cecchini G, Besati C, Ambesi-Impiombato FS, Marchisio PC, De Filippi R - J. Cell Biol. (1998)

Bottom Line: Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted alphavbeta3 enrichment at FCs and impaired adhesion.Accordingly, activation of alphavbeta3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF.These results provide the first example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.

View Article: PubMed Central - PubMed

Affiliation: DIBIT, Department of Biological and Technological Research, San Raffaele Scientific Institute, 20132 Milano, Italy.

ABSTRACT
Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, alphavbeta3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, alphavbeta3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. alphavbeta3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for alphavbeta3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted alphavbeta3 enrichment at FCs and impaired adhesion. Accordingly, activation of alphavbeta3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.

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HGF/SF promotes clustering of β3 integrin at FCs (A  and B). HTU-5 cells were either left untreated (A) or incubated  for 1 h with 50 ng/ml purified HGF/SF (B); cells were then fixed,  permeabilized, and processed for immunofluorescence using an  mAb against β3. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells selectively induces β3 disconnection from  FCs (C-H). HTU-34 cells were incubated for 2 d with either a  preimmune sheep serum (C, E, and G) or with a function-blocking sheep antiserum to HGF/SF (D, F, and H); cells were then  fixed, permeabilized, and processed for immunofluorescence using mAbs against β3 (C and D), β1 (E and F) and vinculin (G and  H). HGF/SF inhibition resulted in strong reduction of the β3  staining at FCs, with partial peripheral redistribution. No modifications of the immunoreactivity for β1 and vinculin could be observed upon HGF/SF blockade. Bar, 10 μm.
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Figure 7: HGF/SF promotes clustering of β3 integrin at FCs (A and B). HTU-5 cells were either left untreated (A) or incubated for 1 h with 50 ng/ml purified HGF/SF (B); cells were then fixed, permeabilized, and processed for immunofluorescence using an mAb against β3. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells selectively induces β3 disconnection from FCs (C-H). HTU-34 cells were incubated for 2 d with either a preimmune sheep serum (C, E, and G) or with a function-blocking sheep antiserum to HGF/SF (D, F, and H); cells were then fixed, permeabilized, and processed for immunofluorescence using mAbs against β3 (C and D), β1 (E and F) and vinculin (G and H). HGF/SF inhibition resulted in strong reduction of the β3 staining at FCs, with partial peripheral redistribution. No modifications of the immunoreactivity for β1 and vinculin could be observed upon HGF/SF blockade. Bar, 10 μm.

Mentions: In immunofluorescence experiments on HTU-5 cells adhering onto endogenous ECM molecules, HGF/SF triggered clustering of αvβ3 at FCs (Fig. 7, A and B). Consistent with this observation, treatment of HTU-34 cells with the inhibitory antibody to HGF/SF resulted in a clear-cut decrease in the focal immunostaining for αvβ3 (Fig. 7, C and D), but no modifications in the topography of the β1 subunit (Fig. 7, E and F) or vinculin (Fig. 7, G and H) were observed. αvβ3 expression levels in HTU-5 clones did not increase upon stimulation with HGF/SF, nor did they decrease after antibody-mediated neutralization of HGF/SF activity in the HTU-34 cultures (not shown).


Growth factor-dependent activation of alphavbeta3 integrin in normal epithelial cells: implications for tumor invasion.

Trusolino L, Serini G, Cecchini G, Besati C, Ambesi-Impiombato FS, Marchisio PC, De Filippi R - J. Cell Biol. (1998)

HGF/SF promotes clustering of β3 integrin at FCs (A  and B). HTU-5 cells were either left untreated (A) or incubated  for 1 h with 50 ng/ml purified HGF/SF (B); cells were then fixed,  permeabilized, and processed for immunofluorescence using an  mAb against β3. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells selectively induces β3 disconnection from  FCs (C-H). HTU-34 cells were incubated for 2 d with either a  preimmune sheep serum (C, E, and G) or with a function-blocking sheep antiserum to HGF/SF (D, F, and H); cells were then  fixed, permeabilized, and processed for immunofluorescence using mAbs against β3 (C and D), β1 (E and F) and vinculin (G and  H). HGF/SF inhibition resulted in strong reduction of the β3  staining at FCs, with partial peripheral redistribution. No modifications of the immunoreactivity for β1 and vinculin could be observed upon HGF/SF blockade. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132885&req=5

Figure 7: HGF/SF promotes clustering of β3 integrin at FCs (A and B). HTU-5 cells were either left untreated (A) or incubated for 1 h with 50 ng/ml purified HGF/SF (B); cells were then fixed, permeabilized, and processed for immunofluorescence using an mAb against β3. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells selectively induces β3 disconnection from FCs (C-H). HTU-34 cells were incubated for 2 d with either a preimmune sheep serum (C, E, and G) or with a function-blocking sheep antiserum to HGF/SF (D, F, and H); cells were then fixed, permeabilized, and processed for immunofluorescence using mAbs against β3 (C and D), β1 (E and F) and vinculin (G and H). HGF/SF inhibition resulted in strong reduction of the β3 staining at FCs, with partial peripheral redistribution. No modifications of the immunoreactivity for β1 and vinculin could be observed upon HGF/SF blockade. Bar, 10 μm.
Mentions: In immunofluorescence experiments on HTU-5 cells adhering onto endogenous ECM molecules, HGF/SF triggered clustering of αvβ3 at FCs (Fig. 7, A and B). Consistent with this observation, treatment of HTU-34 cells with the inhibitory antibody to HGF/SF resulted in a clear-cut decrease in the focal immunostaining for αvβ3 (Fig. 7, C and D), but no modifications in the topography of the β1 subunit (Fig. 7, E and F) or vinculin (Fig. 7, G and H) were observed. αvβ3 expression levels in HTU-5 clones did not increase upon stimulation with HGF/SF, nor did they decrease after antibody-mediated neutralization of HGF/SF activity in the HTU-34 cultures (not shown).

Bottom Line: Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted alphavbeta3 enrichment at FCs and impaired adhesion.Accordingly, activation of alphavbeta3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF.These results provide the first example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.

View Article: PubMed Central - PubMed

Affiliation: DIBIT, Department of Biological and Technological Research, San Raffaele Scientific Institute, 20132 Milano, Italy.

ABSTRACT
Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, alphavbeta3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, alphavbeta3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. alphavbeta3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for alphavbeta3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted alphavbeta3 enrichment at FCs and impaired adhesion. Accordingly, activation of alphavbeta3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.

Show MeSH
Related in: MedlinePlus