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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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A soluble factor produced by HTU-34 cells is responsible for αvβ3 ligand-binding activity and recruitment to FCs. (A)  Adhesion assay. Treatment of HTU-34 cells (open bars) with  suramin (Sur) markedly reduced adhesion to VN. Conversely,  conditioned medium from the HTU-34 clone (Cond.) triggered  adhesion of HTU-5 cells (hatched bars). (B and C) Immunofluorescence. HTU-5 cells were either left untreated (B) or stimulated for 1 h with conditioned medium from the HTU-34 clone  (C); cells were then fixed, permeabilized, and processed for immunofluorescence using an mAb against the β3 integrin subunit.  Bar, 10 μm.
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Figure 5: A soluble factor produced by HTU-34 cells is responsible for αvβ3 ligand-binding activity and recruitment to FCs. (A) Adhesion assay. Treatment of HTU-34 cells (open bars) with suramin (Sur) markedly reduced adhesion to VN. Conversely, conditioned medium from the HTU-34 clone (Cond.) triggered adhesion of HTU-5 cells (hatched bars). (B and C) Immunofluorescence. HTU-5 cells were either left untreated (B) or stimulated for 1 h with conditioned medium from the HTU-34 clone (C); cells were then fixed, permeabilized, and processed for immunofluorescence using an mAb against the β3 integrin subunit. Bar, 10 μm.

Mentions: In a preliminary test of this possibility we plated HTU-34 cells on VN after preincubation with suramin, a drug that blocks any cytokine–receptor interactions (La Rocca et al., 1990; Adams et al., 1991; Ferracini et al., 1995; Zumkeller and Schofield, 1995). Indeed, HTU-34 cells completely lost their adhesion potential (Fig. 5 A) suggesting that αvβ3 adhesive properties were controlled by a soluble factor interacting with a receptor. To test this hypothesis we challenged HTU-5 cells with SFM conditioned by the HTU-34 clone and found that cells acquired de novo adhesion to VN (Fig. 5 A). When HTU-34–conditioned medium was applied to HTU-5 cells previously plated onto glass coverslips, thus adhering to endogenous ECM molecules, αvβ3 recruitment at FCs was observed (Fig. 5, B and C). It was deduced that a soluble factor produced by malignant cells, but not by normal cells, controlled αvβ3-mediated adhesion by acting on a receptor shared by the two cell types.


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 soluble factor produced by HTU-34 cells is responsible for αvβ3 ligand-binding activity and recruitment to FCs. (A)  Adhesion assay. Treatment of HTU-34 cells (open bars) with  suramin (Sur) markedly reduced adhesion to VN. Conversely,  conditioned medium from the HTU-34 clone (Cond.) triggered  adhesion of HTU-5 cells (hatched bars). (B and C) Immunofluorescence. HTU-5 cells were either left untreated (B) or stimulated for 1 h with conditioned medium from the HTU-34 clone  (C); cells were then fixed, permeabilized, and processed for immunofluorescence using an mAb against the β3 integrin subunit.  Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132885&req=5

Figure 5: A soluble factor produced by HTU-34 cells is responsible for αvβ3 ligand-binding activity and recruitment to FCs. (A) Adhesion assay. Treatment of HTU-34 cells (open bars) with suramin (Sur) markedly reduced adhesion to VN. Conversely, conditioned medium from the HTU-34 clone (Cond.) triggered adhesion of HTU-5 cells (hatched bars). (B and C) Immunofluorescence. HTU-5 cells were either left untreated (B) or stimulated for 1 h with conditioned medium from the HTU-34 clone (C); cells were then fixed, permeabilized, and processed for immunofluorescence using an mAb against the β3 integrin subunit. Bar, 10 μm.
Mentions: In a preliminary test of this possibility we plated HTU-34 cells on VN after preincubation with suramin, a drug that blocks any cytokine–receptor interactions (La Rocca et al., 1990; Adams et al., 1991; Ferracini et al., 1995; Zumkeller and Schofield, 1995). Indeed, HTU-34 cells completely lost their adhesion potential (Fig. 5 A) suggesting that αvβ3 adhesive properties were controlled by a soluble factor interacting with a receptor. To test this hypothesis we challenged HTU-5 cells with SFM conditioned by the HTU-34 clone and found that cells acquired de novo adhesion to VN (Fig. 5 A). When HTU-34–conditioned medium was applied to HTU-5 cells previously plated onto glass coverslips, thus adhering to endogenous ECM molecules, αvβ3 recruitment at FCs was observed (Fig. 5, B and C). It was deduced that a soluble factor produced by malignant cells, but not by normal cells, controlled αvβ3-mediated adhesion by acting on a receptor shared by the two cell types.

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