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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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αvβ3 integrin displays ligand-binding activity and is recruited to newly forming adhesive structures only in HTU-34  cells. (A–D) Adhesion assays onto αvβ3-specific ECM molecules.  (A–C) Cells from HTU-5 (closed symbols) and HTU-34 (open  symbols) cultures were detached, resuspended in SFM, and then  plated onto microtiter wells coated with increasing amounts of  VN (A), fibrinogen (B), and osteopontin (C). After allowing cells  to attach, the extent of cell adhesion was quantitated as described  in Materials and Methods. (D) Adhesion inhibition assay.  HTU-34 cells were plated onto 10 μg/ml VN alone (−) or in the  presence of increasing concentrations of function-blocking mAbs  to αvβ3 (LM 609) or to β1 (AII B2). (E–G) Immunofluorescence. HTU-34 cells were detached, resuspended in SFM, and  plated for 2 h onto glass coverslips coated with VN. Cells were  then fixed, permeabilized, and processed for double immunofluorescence using a rabbit polyclonal antiserum against β3 (E) and  an mAb against vinculin (F). An mAb against β1 was also used  (G) in single immunofluorescence experiments. In adhering  HTU-34 cells, β3 and vinculin, but not β1, were recruited to maturing FCs. Bar, 7 μm.
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Figure 3: αvβ3 integrin displays ligand-binding activity and is recruited to newly forming adhesive structures only in HTU-34 cells. (A–D) Adhesion assays onto αvβ3-specific ECM molecules. (A–C) Cells from HTU-5 (closed symbols) and HTU-34 (open symbols) cultures were detached, resuspended in SFM, and then plated onto microtiter wells coated with increasing amounts of VN (A), fibrinogen (B), and osteopontin (C). After allowing cells to attach, the extent of cell adhesion was quantitated as described in Materials and Methods. (D) Adhesion inhibition assay. HTU-34 cells were plated onto 10 μg/ml VN alone (−) or in the presence of increasing concentrations of function-blocking mAbs to αvβ3 (LM 609) or to β1 (AII B2). (E–G) Immunofluorescence. HTU-34 cells were detached, resuspended in SFM, and plated for 2 h onto glass coverslips coated with VN. Cells were then fixed, permeabilized, and processed for double immunofluorescence using a rabbit polyclonal antiserum against β3 (E) and an mAb against vinculin (F). An mAb against β1 was also used (G) in single immunofluorescence experiments. In adhering HTU-34 cells, β3 and vinculin, but not β1, were recruited to maturing FCs. Bar, 7 μm.

Mentions: To induce ligand-mediated clustering of αvβ3 in HTU-5 normal cells, subconfluent cultures were detached and plated in serum-free conditions onto a plastic substratum coated with a concentration range (2.5 to 25 μg/ml) of VN (Fig. 3 A), fibrinogen (Fig. 3 B), and osteopontin (Fig. 3 C). Surprisingly, HTU-5 cells could not attach and spread. In contrast, HTU-34 cells rapidly adhered and firmly spread; indeed, cells attached proportionally to the amount of substratum and were detectable even at very low doses of matrix ligands (Fig. 3, A–C). HTU-34 cell attachment and spreading on VN was progressively impaired by adding increasing concentrations of the αvβ3 inhibitory mAb LM609 but not by the β1 function-blocking mAb AIIB2 (Fig. 3 D). Moreover, in HTU-34 cells plated on VN and processed for immunofluorescence after fixation and permeabilization, the only integrin receptor clustered at nascent FC, strictly colocalizing with vinculin (Fig. 3 F), was αvβ3 (Fig. 3 E). Conversely, β1 integrins were almost undetectable on the cell surface (Fig. 3 G). Thus, adhesion of HTU-34 cells to VN was specifically mediated by αvβ3.


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)

αvβ3 integrin displays ligand-binding activity and is recruited to newly forming adhesive structures only in HTU-34  cells. (A–D) Adhesion assays onto αvβ3-specific ECM molecules.  (A–C) Cells from HTU-5 (closed symbols) and HTU-34 (open  symbols) cultures were detached, resuspended in SFM, and then  plated onto microtiter wells coated with increasing amounts of  VN (A), fibrinogen (B), and osteopontin (C). After allowing cells  to attach, the extent of cell adhesion was quantitated as described  in Materials and Methods. (D) Adhesion inhibition assay.  HTU-34 cells were plated onto 10 μg/ml VN alone (−) or in the  presence of increasing concentrations of function-blocking mAbs  to αvβ3 (LM 609) or to β1 (AII B2). (E–G) Immunofluorescence. HTU-34 cells were detached, resuspended in SFM, and  plated for 2 h onto glass coverslips coated with VN. Cells were  then fixed, permeabilized, and processed for double immunofluorescence using a rabbit polyclonal antiserum against β3 (E) and  an mAb against vinculin (F). An mAb against β1 was also used  (G) in single immunofluorescence experiments. In adhering  HTU-34 cells, β3 and vinculin, but not β1, were recruited to maturing FCs. Bar, 7 μm.
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Figure 3: αvβ3 integrin displays ligand-binding activity and is recruited to newly forming adhesive structures only in HTU-34 cells. (A–D) Adhesion assays onto αvβ3-specific ECM molecules. (A–C) Cells from HTU-5 (closed symbols) and HTU-34 (open symbols) cultures were detached, resuspended in SFM, and then plated onto microtiter wells coated with increasing amounts of VN (A), fibrinogen (B), and osteopontin (C). After allowing cells to attach, the extent of cell adhesion was quantitated as described in Materials and Methods. (D) Adhesion inhibition assay. HTU-34 cells were plated onto 10 μg/ml VN alone (−) or in the presence of increasing concentrations of function-blocking mAbs to αvβ3 (LM 609) or to β1 (AII B2). (E–G) Immunofluorescence. HTU-34 cells were detached, resuspended in SFM, and plated for 2 h onto glass coverslips coated with VN. Cells were then fixed, permeabilized, and processed for double immunofluorescence using a rabbit polyclonal antiserum against β3 (E) and an mAb against vinculin (F). An mAb against β1 was also used (G) in single immunofluorescence experiments. In adhering HTU-34 cells, β3 and vinculin, but not β1, were recruited to maturing FCs. Bar, 7 μm.
Mentions: To induce ligand-mediated clustering of αvβ3 in HTU-5 normal cells, subconfluent cultures were detached and plated in serum-free conditions onto a plastic substratum coated with a concentration range (2.5 to 25 μg/ml) of VN (Fig. 3 A), fibrinogen (Fig. 3 B), and osteopontin (Fig. 3 C). Surprisingly, HTU-5 cells could not attach and spread. In contrast, HTU-34 cells rapidly adhered and firmly spread; indeed, cells attached proportionally to the amount of substratum and were detectable even at very low doses of matrix ligands (Fig. 3, A–C). HTU-34 cell attachment and spreading on VN was progressively impaired by adding increasing concentrations of the αvβ3 inhibitory mAb LM609 but not by the β1 function-blocking mAb AIIB2 (Fig. 3 D). Moreover, in HTU-34 cells plated on VN and processed for immunofluorescence after fixation and permeabilization, the only integrin receptor clustered at nascent FC, strictly colocalizing with vinculin (Fig. 3 F), was αvβ3 (Fig. 3 E). Conversely, β1 integrins were almost undetectable on the cell surface (Fig. 3 G). Thus, adhesion of HTU-34 cells to VN was specifically mediated by αvβ3.

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