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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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β3 integrin is clustered at FCs only in HTU-34 cells. In  A and B, HTU-5 (A) and HTU-34 (B) cells were fixed and directly immunostained with a β3 mAb. In C and F, HTU-5 (C and  D) and HTU-34 (E and F) cells were fixed, permeabilized with a  Triton X-100 buffer that removes most of the soluble proteins but  not cytoskeleton-associated molecules, and then stained by a  double immunofluorescence protocol using the β3 mAb followed  by a rhodamine-conjugated rabbit anti–mouse IgG (C and E) and  by fluorescein-tagged phalloidin (D and F). In nonpermeabilized  cells, β3 displayed a fine grainy pattern (A and B) and the signal  was much stronger in the HTU-34 strain (B). After permeabilization, β3 immunoreactivity was completely abrogated in HTU-5  cells (C and D) and selectively enriched at FCs in the HTU-34  clone (E and F). Bar, 10 μm.
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Figure 2: β3 integrin is clustered at FCs only in HTU-34 cells. In A and B, HTU-5 (A) and HTU-34 (B) cells were fixed and directly immunostained with a β3 mAb. In C and F, HTU-5 (C and D) and HTU-34 (E and F) cells were fixed, permeabilized with a Triton X-100 buffer that removes most of the soluble proteins but not cytoskeleton-associated molecules, and then stained by a double immunofluorescence protocol using the β3 mAb followed by a rhodamine-conjugated rabbit anti–mouse IgG (C and E) and by fluorescein-tagged phalloidin (D and F). In nonpermeabilized cells, β3 displayed a fine grainy pattern (A and B) and the signal was much stronger in the HTU-34 strain (B). After permeabilization, β3 immunoreactivity was completely abrogated in HTU-5 cells (C and D) and selectively enriched at FCs in the HTU-34 clone (E and F). Bar, 10 μm.

Mentions: The only modification in integrin repertoire observed in malignant versus normal thyroid cells was the higher surface expression of the αvβ3 heterodimer in HTU-34 cells (Fig. 1, A–D). To evaluate the subcellular distribution of αvβ3 in normal and carcinoma clones, cells were plated onto glass coverslips, cultured for several days, and then subjected to immunofluorescence. Under these conditions, cell adhesion occurs because of endogenous production of matrix molecules. Immunofluorescence experiments on fixed, nonpermeabilized cells were in accordance with the immunoprecipitation data: a fine grainy pattern of immunoreactivity was much stronger in HTU-34 (Fig. 2 B) than in HTU-5 cells (Fig. 2 A). Interestingly, treatment of fixed cells with permeabilization buffer (0.5% Triton X-100), which extracts freely diffusing molecules yet preserves actin cytoskeletal connections (Fey et al., 1983; Rabinovitz and Mercurio, 1997), completely removed β3 immunoreactivity in HTU-5 cells (Fig. 2, C and D) and selectively concentrated the β3 fluorescent signal of HTU-34 cells at the endings of microfilament bundles in sites compatible with FCs (Fig. 2, E and F). The same result was obtained when Triton X-100 permeabilization was performed before fixation (not shown). Thus, recruitment of the αvβ3 heterodimer to microfilament-associated adhesion sites occurs only in malignant but not in normal thyroid 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)

β3 integrin is clustered at FCs only in HTU-34 cells. In  A and B, HTU-5 (A) and HTU-34 (B) cells were fixed and directly immunostained with a β3 mAb. In C and F, HTU-5 (C and  D) and HTU-34 (E and F) cells were fixed, permeabilized with a  Triton X-100 buffer that removes most of the soluble proteins but  not cytoskeleton-associated molecules, and then stained by a  double immunofluorescence protocol using the β3 mAb followed  by a rhodamine-conjugated rabbit anti–mouse IgG (C and E) and  by fluorescein-tagged phalloidin (D and F). In nonpermeabilized  cells, β3 displayed a fine grainy pattern (A and B) and the signal  was much stronger in the HTU-34 strain (B). After permeabilization, β3 immunoreactivity was completely abrogated in HTU-5  cells (C and D) and selectively enriched at FCs in the HTU-34  clone (E and F). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 2: β3 integrin is clustered at FCs only in HTU-34 cells. In A and B, HTU-5 (A) and HTU-34 (B) cells were fixed and directly immunostained with a β3 mAb. In C and F, HTU-5 (C and D) and HTU-34 (E and F) cells were fixed, permeabilized with a Triton X-100 buffer that removes most of the soluble proteins but not cytoskeleton-associated molecules, and then stained by a double immunofluorescence protocol using the β3 mAb followed by a rhodamine-conjugated rabbit anti–mouse IgG (C and E) and by fluorescein-tagged phalloidin (D and F). In nonpermeabilized cells, β3 displayed a fine grainy pattern (A and B) and the signal was much stronger in the HTU-34 strain (B). After permeabilization, β3 immunoreactivity was completely abrogated in HTU-5 cells (C and D) and selectively enriched at FCs in the HTU-34 clone (E and F). Bar, 10 μm.
Mentions: The only modification in integrin repertoire observed in malignant versus normal thyroid cells was the higher surface expression of the αvβ3 heterodimer in HTU-34 cells (Fig. 1, A–D). To evaluate the subcellular distribution of αvβ3 in normal and carcinoma clones, cells were plated onto glass coverslips, cultured for several days, and then subjected to immunofluorescence. Under these conditions, cell adhesion occurs because of endogenous production of matrix molecules. Immunofluorescence experiments on fixed, nonpermeabilized cells were in accordance with the immunoprecipitation data: a fine grainy pattern of immunoreactivity was much stronger in HTU-34 (Fig. 2 B) than in HTU-5 cells (Fig. 2 A). Interestingly, treatment of fixed cells with permeabilization buffer (0.5% Triton X-100), which extracts freely diffusing molecules yet preserves actin cytoskeletal connections (Fey et al., 1983; Rabinovitz and Mercurio, 1997), completely removed β3 immunoreactivity in HTU-5 cells (Fig. 2, C and D) and selectively concentrated the β3 fluorescent signal of HTU-34 cells at the endings of microfilament bundles in sites compatible with FCs (Fig. 2, E and F). The same result was obtained when Triton X-100 permeabilization was performed before fixation (not shown). Thus, recruitment of the αvβ3 heterodimer to microfilament-associated adhesion sites occurs only in malignant but not in normal thyroid 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