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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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Biochemical evidence of HGF/SF autocrine production  by the HTU-34 clone. HTU-5 cells were either left untreated or  incubated for 10 min with conditioned medium from the HTU-34  clone (cm HTU-5) or with 50 ng purified HGF/SF (HGF/SF  HTU-5). Unstimulated and stimulated HTU-5 cells, together  with untreated HTU-34 cells, were extracted and immunoprecipitated with a human c-Met polyclonal antibody. The eluates were  then split into two equal fractions, Western blotted, and decorated with mAbs to c-Met (A, anti-Met) or to phosphotyrosine (B,  anti-PY). The c-Met receptor β chain (Met145β) appeared to be  constitutively tyrosine-phosphorylated in the HTU-34 strain.  Specific tyrosine-phosphorylation of the Met protein could be induced in HTU-5 cells by treatment with HTU-34–conditioned  medium or with HGF/SF. The HGF/SF transcript is specifically  expressed in HTU-34, but not in HTU-5, cells (C). 30 μg of total  RNA from HTU-5 and HTU-34 cells were separated by electrophoresis, transferred to nylon filters, and hybridized to 32P- labeled probes specific for HGF/SF and for the housekeeper  gene GAPDH.
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Figure 8: Biochemical evidence of HGF/SF autocrine production by the HTU-34 clone. HTU-5 cells were either left untreated or incubated for 10 min with conditioned medium from the HTU-34 clone (cm HTU-5) or with 50 ng purified HGF/SF (HGF/SF HTU-5). Unstimulated and stimulated HTU-5 cells, together with untreated HTU-34 cells, were extracted and immunoprecipitated with a human c-Met polyclonal antibody. The eluates were then split into two equal fractions, Western blotted, and decorated with mAbs to c-Met (A, anti-Met) or to phosphotyrosine (B, anti-PY). The c-Met receptor β chain (Met145β) appeared to be constitutively tyrosine-phosphorylated in the HTU-34 strain. Specific tyrosine-phosphorylation of the Met protein could be induced in HTU-5 cells by treatment with HTU-34–conditioned medium or with HGF/SF. The HGF/SF transcript is specifically expressed in HTU-34, but not in HTU-5, cells (C). 30 μg of total RNA from HTU-5 and HTU-34 cells were separated by electrophoresis, transferred to nylon filters, and hybridized to 32P- labeled probes specific for HGF/SF and for the housekeeper gene GAPDH.

Mentions: To determine whether HTU-5 and HTU-34 cells expressed the HGF/SF receptor c-Met and to verify whether the receptor was constitutively activated in HTU-34 cells because of a chronic autocrine loop, cell lysates were subjected to immunoprecipitation with the C-28 human Met polyclonal antibody. Anti-Met immunoprecipitates were then split into two equal fractions, Western blotted, and decorated with the anti-Met mAb DQ-13 (Fig. 8 A) or the phosphotyrosine mAb 4G10 (Fig. 8 B). In both cell lines, the 145-kD mature form of the c-Met β subunit was clearly detected (Fig. 8 A). The c-Met β subunit was phosphorylated on tyrosine residues in HTU-34 cell extracts, but not in unstimulated HTU-5 cell lysates. When HTU-5 cells were treated with conditioned medium from the HTU-34 clone or with purified HGF/SF, specific tyrosine phosphorylation of the c-Met β subunit was detected (Fig. 8 B).


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)

Biochemical evidence of HGF/SF autocrine production  by the HTU-34 clone. HTU-5 cells were either left untreated or  incubated for 10 min with conditioned medium from the HTU-34  clone (cm HTU-5) or with 50 ng purified HGF/SF (HGF/SF  HTU-5). Unstimulated and stimulated HTU-5 cells, together  with untreated HTU-34 cells, were extracted and immunoprecipitated with a human c-Met polyclonal antibody. The eluates were  then split into two equal fractions, Western blotted, and decorated with mAbs to c-Met (A, anti-Met) or to phosphotyrosine (B,  anti-PY). The c-Met receptor β chain (Met145β) appeared to be  constitutively tyrosine-phosphorylated in the HTU-34 strain.  Specific tyrosine-phosphorylation of the Met protein could be induced in HTU-5 cells by treatment with HTU-34–conditioned  medium or with HGF/SF. The HGF/SF transcript is specifically  expressed in HTU-34, but not in HTU-5, cells (C). 30 μg of total  RNA from HTU-5 and HTU-34 cells were separated by electrophoresis, transferred to nylon filters, and hybridized to 32P- labeled probes specific for HGF/SF and for the housekeeper  gene GAPDH.
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Related In: Results  -  Collection

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Figure 8: Biochemical evidence of HGF/SF autocrine production by the HTU-34 clone. HTU-5 cells were either left untreated or incubated for 10 min with conditioned medium from the HTU-34 clone (cm HTU-5) or with 50 ng purified HGF/SF (HGF/SF HTU-5). Unstimulated and stimulated HTU-5 cells, together with untreated HTU-34 cells, were extracted and immunoprecipitated with a human c-Met polyclonal antibody. The eluates were then split into two equal fractions, Western blotted, and decorated with mAbs to c-Met (A, anti-Met) or to phosphotyrosine (B, anti-PY). The c-Met receptor β chain (Met145β) appeared to be constitutively tyrosine-phosphorylated in the HTU-34 strain. Specific tyrosine-phosphorylation of the Met protein could be induced in HTU-5 cells by treatment with HTU-34–conditioned medium or with HGF/SF. The HGF/SF transcript is specifically expressed in HTU-34, but not in HTU-5, cells (C). 30 μg of total RNA from HTU-5 and HTU-34 cells were separated by electrophoresis, transferred to nylon filters, and hybridized to 32P- labeled probes specific for HGF/SF and for the housekeeper gene GAPDH.
Mentions: To determine whether HTU-5 and HTU-34 cells expressed the HGF/SF receptor c-Met and to verify whether the receptor was constitutively activated in HTU-34 cells because of a chronic autocrine loop, cell lysates were subjected to immunoprecipitation with the C-28 human Met polyclonal antibody. Anti-Met immunoprecipitates were then split into two equal fractions, Western blotted, and decorated with the anti-Met mAb DQ-13 (Fig. 8 A) or the phosphotyrosine mAb 4G10 (Fig. 8 B). In both cell lines, the 145-kD mature form of the c-Met β subunit was clearly detected (Fig. 8 A). The c-Met β subunit was phosphorylated on tyrosine residues in HTU-34 cell extracts, but not in unstimulated HTU-5 cell lysates. When HTU-5 cells were treated with conditioned medium from the HTU-34 clone or with purified HGF/SF, specific tyrosine phosphorylation of the c-Met β subunit was detected (Fig. 8 B).

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