Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Adhesion and adhesion inhibition assay of HTU-5  (hatched bars) and HTU-34 (open bars) cells onto FN. Cells were  plated onto 10 μg/ml FN alone (−) or in the presence of mAbs  LM609 (10 μg/ml) and/or AIIB2 (1:10 dilution). (B–E) Immunofluorescence. When HTU-5 cells were allowed to adhere to FN  (B and C), the β1 chain (B) but not the β3 subunit (C) was clustered at nascent FCs. In HTU-34 cells plated on FN, both integrins were recruited to adhesion sites (D and E). Double-immunofluorescence analysis using an mAb to β1 (D) and a rabbit  polyclonal antiserum to β3 (E) showed that both subunits were  clustered within the same focal adhesions. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132885&req=5

Figure 4: (A) Adhesion and adhesion inhibition assay of HTU-5 (hatched bars) and HTU-34 (open bars) cells onto FN. Cells were plated onto 10 μg/ml FN alone (−) or in the presence of mAbs LM609 (10 μg/ml) and/or AIIB2 (1:10 dilution). (B–E) Immunofluorescence. When HTU-5 cells were allowed to adhere to FN (B and C), the β1 chain (B) but not the β3 subunit (C) was clustered at nascent FCs. In HTU-34 cells plated on FN, both integrins were recruited to adhesion sites (D and E). Double-immunofluorescence analysis using an mAb to β1 (D) and a rabbit polyclonal antiserum to β3 (E) showed that both subunits were clustered within the same focal adhesions. Bar, 10 μm.

Mentions: When HTU-5 were plated onto FN, a ligand for both αvβ3 and αvβ1, cells could attach and spread. In this case as well, αvβ3 was not involved in the adhesive phenomenon: only the β1 inhibitory mAb efficiently blocked adhesion whereas mAb LM609 did not display any significant effect (Fig. 4 A). Conversely, β1 and β3 integrins were equally responsible for adhesion to FN in HTU-34 cells: function-blocking mAbs against either integrins could partially impair adhesion when added individually, and almost totally when added together (Fig. 4 A). Immunofluorescence experiments showed that HTU-5 cells, when plated on FN, organized β1 integrins at adhesive structures (Fig. 4 B), whereas αvβ3 was almost undetectable (Fig. 4 C). On the contrary, in HTU-34 plated on FN both β1 and β3 integrins were highly enriched at focal adhesions; double immunostaining for β1 and β3 revealed colocalization of the two integrin subunits within the same FCs (Fig. 4, D and E).


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)

(A) Adhesion and adhesion inhibition assay of HTU-5  (hatched bars) and HTU-34 (open bars) cells onto FN. Cells were  plated onto 10 μg/ml FN alone (−) or in the presence of mAbs  LM609 (10 μg/ml) and/or AIIB2 (1:10 dilution). (B–E) Immunofluorescence. When HTU-5 cells were allowed to adhere to FN  (B and C), the β1 chain (B) but not the β3 subunit (C) was clustered at nascent FCs. In HTU-34 cells plated on FN, both integrins were recruited to adhesion sites (D and E). Double-immunofluorescence analysis using an mAb to β1 (D) and a rabbit  polyclonal antiserum to β3 (E) showed that both subunits were  clustered within the same focal adhesions. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: (A) Adhesion and adhesion inhibition assay of HTU-5 (hatched bars) and HTU-34 (open bars) cells onto FN. Cells were plated onto 10 μg/ml FN alone (−) or in the presence of mAbs LM609 (10 μg/ml) and/or AIIB2 (1:10 dilution). (B–E) Immunofluorescence. When HTU-5 cells were allowed to adhere to FN (B and C), the β1 chain (B) but not the β3 subunit (C) was clustered at nascent FCs. In HTU-34 cells plated on FN, both integrins were recruited to adhesion sites (D and E). Double-immunofluorescence analysis using an mAb to β1 (D) and a rabbit polyclonal antiserum to β3 (E) showed that both subunits were clustered within the same focal adhesions. Bar, 10 μm.
Mentions: When HTU-5 were plated onto FN, a ligand for both αvβ3 and αvβ1, cells could attach and spread. In this case as well, αvβ3 was not involved in the adhesive phenomenon: only the β1 inhibitory mAb efficiently blocked adhesion whereas mAb LM609 did not display any significant effect (Fig. 4 A). Conversely, β1 and β3 integrins were equally responsible for adhesion to FN in HTU-34 cells: function-blocking mAbs against either integrins could partially impair adhesion when added individually, and almost totally when added together (Fig. 4 A). Immunofluorescence experiments showed that HTU-5 cells, when plated on FN, organized β1 integrins at adhesive structures (Fig. 4 B), whereas αvβ3 was almost undetectable (Fig. 4 C). On the contrary, in HTU-34 plated on FN both β1 and β3 integrins were highly enriched at focal adhesions; double immunostaining for β1 and β3 revealed colocalization of the two integrin subunits within the same FCs (Fig. 4, D and E).

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