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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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Characterization of the surface integrin repertoire in HTU-5 and  HTU-34 thyroid clonal strains. Confluent cells were surface biotinylated and  detergent lysates were immunoprecipitated with the indicated mAbs to different integrin subunits as described in  Materials and Methods. The eluates  were then analyzed by SDS-PAGE under nonreducing conditions. To demonstrate association of the β1 subunit  with the αv chain, anti-αv immunoprecipitates were Western blotted and then decorated with a β1 polyclonal  antiserum (E). Cell surface expression of the β3 subunit was also assessed by FACS® analysis (C and D). Washed, unfixed HTU-5 (C) and  HTU-34 (D) cells were stained for indirect immunofluorescence and  flow cytometry analysis as described in Materials and Methods. Each  profile was generated from analyzing 10,000 cells. Relative values of  MIF were derived from gated computerized histogram analysis and expressed as log arbitrary units. Nonspecific fluorescence was measured  using the secondary fluorescein-tagged rabbit anti–mouse IgGs alone  (shaded diagrams) and found to have a MIF <7. Specific β3 fluorescence (black diagrams) corresponded to a MIF of 38.7 in HTU-5 cells  and 238.44 in HTU-34 cells.
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Figure 1: Characterization of the surface integrin repertoire in HTU-5 and HTU-34 thyroid clonal strains. Confluent cells were surface biotinylated and detergent lysates were immunoprecipitated with the indicated mAbs to different integrin subunits as described in Materials and Methods. The eluates were then analyzed by SDS-PAGE under nonreducing conditions. To demonstrate association of the β1 subunit with the αv chain, anti-αv immunoprecipitates were Western blotted and then decorated with a β1 polyclonal antiserum (E). Cell surface expression of the β3 subunit was also assessed by FACS® analysis (C and D). Washed, unfixed HTU-5 (C) and HTU-34 (D) cells were stained for indirect immunofluorescence and flow cytometry analysis as described in Materials and Methods. Each profile was generated from analyzing 10,000 cells. Relative values of MIF were derived from gated computerized histogram analysis and expressed as log arbitrary units. Nonspecific fluorescence was measured using the secondary fluorescein-tagged rabbit anti–mouse IgGs alone (shaded diagrams) and found to have a MIF <7. Specific β3 fluorescence (black diagrams) corresponded to a MIF of 38.7 in HTU-5 cells and 238.44 in HTU-34 cells.

Mentions: To characterize the surface adhesive repertoire of normal (HTU-5) and malignant (HTU-34) thyroid cells, a battery of integrin-specific mAbs was used in immunoprecipitation experiments on membrane biotinylated cell monolayers. The αv mAb L230 immunoprecipitated three bands of 150, 130, and 90 kD in both the normal and carcinoma strains (Fig. 1 A). The 150/90-kD doublet was also brought down by the β3-specific mAb VIPL2 (Fig. 1 B) but not by the β5 mAb IA9 (not shown). Based on band intensities, the αvβ3 integrin appeared to be more expressed in the HTU-34 clone. The higher surface exposure of αvβ3 in carcinoma cells was confirmed by FACS® analysis on HTU-5 (Fig. 1 C) and HTU-34 (Fig. 1 D) cells.


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)

Characterization of the surface integrin repertoire in HTU-5 and  HTU-34 thyroid clonal strains. Confluent cells were surface biotinylated and  detergent lysates were immunoprecipitated with the indicated mAbs to different integrin subunits as described in  Materials and Methods. The eluates  were then analyzed by SDS-PAGE under nonreducing conditions. To demonstrate association of the β1 subunit  with the αv chain, anti-αv immunoprecipitates were Western blotted and then decorated with a β1 polyclonal  antiserum (E). Cell surface expression of the β3 subunit was also assessed by FACS® analysis (C and D). Washed, unfixed HTU-5 (C) and  HTU-34 (D) cells were stained for indirect immunofluorescence and  flow cytometry analysis as described in Materials and Methods. Each  profile was generated from analyzing 10,000 cells. Relative values of  MIF were derived from gated computerized histogram analysis and expressed as log arbitrary units. Nonspecific fluorescence was measured  using the secondary fluorescein-tagged rabbit anti–mouse IgGs alone  (shaded diagrams) and found to have a MIF <7. Specific β3 fluorescence (black diagrams) corresponded to a MIF of 38.7 in HTU-5 cells  and 238.44 in HTU-34 cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132885&req=5

Figure 1: Characterization of the surface integrin repertoire in HTU-5 and HTU-34 thyroid clonal strains. Confluent cells were surface biotinylated and detergent lysates were immunoprecipitated with the indicated mAbs to different integrin subunits as described in Materials and Methods. The eluates were then analyzed by SDS-PAGE under nonreducing conditions. To demonstrate association of the β1 subunit with the αv chain, anti-αv immunoprecipitates were Western blotted and then decorated with a β1 polyclonal antiserum (E). Cell surface expression of the β3 subunit was also assessed by FACS® analysis (C and D). Washed, unfixed HTU-5 (C) and HTU-34 (D) cells were stained for indirect immunofluorescence and flow cytometry analysis as described in Materials and Methods. Each profile was generated from analyzing 10,000 cells. Relative values of MIF were derived from gated computerized histogram analysis and expressed as log arbitrary units. Nonspecific fluorescence was measured using the secondary fluorescein-tagged rabbit anti–mouse IgGs alone (shaded diagrams) and found to have a MIF <7. Specific β3 fluorescence (black diagrams) corresponded to a MIF of 38.7 in HTU-5 cells and 238.44 in HTU-34 cells.
Mentions: To characterize the surface adhesive repertoire of normal (HTU-5) and malignant (HTU-34) thyroid cells, a battery of integrin-specific mAbs was used in immunoprecipitation experiments on membrane biotinylated cell monolayers. The αv mAb L230 immunoprecipitated three bands of 150, 130, and 90 kD in both the normal and carcinoma strains (Fig. 1 A). The 150/90-kD doublet was also brought down by the β3-specific mAb VIPL2 (Fig. 1 B) but not by the β5 mAb IA9 (not shown). Based on band intensities, the αvβ3 integrin appeared to be more expressed in the HTU-34 clone. The higher surface exposure of αvβ3 in carcinoma cells was confirmed by FACS® analysis on HTU-5 (Fig. 1 C) and HTU-34 (Fig. 1 D) cells.

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