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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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HGF/SF selectively  activates the adhesive potential of αvβ3 integrin in  HTU-5 cells. (A) HGF/SF  promotes αvβ3-mediated  adhesion to VN in a dose-dependent manner. Cells  from HTU-5 cultures were  detached, resuspended in  SFM containing increasing  amounts of HGF/SF, and  then plated onto VN. (B)  The effect of HGF/SF on  αvβ3-mediated adhesion is  specific and in malignant  cells it results from an autocrine loop. HGF/SF was the  only stimulus able to trigger  adhesion of HTU-5 cells  (hatched bars) to VN. Treatment of HTU-34 cells (open  bars) with a blocking polyclonal antiserum to HGF/SF  (anti-HGF) markedly reduced adhesion. HGF/SF-induced adhesion in HTU-5  cells was inhibited by mAb  LM609 to the β3 subunit  (HGF+LM609) and by treating HTU-34–conditioned medium with the inhibitory antiserum against HGF/SF (Cond + anti-HGF). In  contrast, addition of preimmune sheep IgGs to HTU-34 cells (P-I) did not modify their adhesive properties. Similarly, treatment of  HTU-5 cells with HTU-34–conditioned medium in the presence of preimmune IgGs (Cond + P-I) had no effect on the ability of conditioned medium to trigger cell adhesion. (C) HGF/SF increases adhesion efficiency of HTU-5 cells on FN via the enhancement of αvβ3  ligand-binding ability (see Results). (D) HGF/SF does not modify the adhesive function of laminin-binding integrins. Attachment of  both HTU-5 (hatched bars) and HTU-34 (open bars) cells to laminin was inhibited by the β1 function-blocking mAb AIIB2, but was not  modulated by HGF/SF treatment.
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Figure 6: HGF/SF selectively activates the adhesive potential of αvβ3 integrin in HTU-5 cells. (A) HGF/SF promotes αvβ3-mediated adhesion to VN in a dose-dependent manner. Cells from HTU-5 cultures were detached, resuspended in SFM containing increasing amounts of HGF/SF, and then plated onto VN. (B) The effect of HGF/SF on αvβ3-mediated adhesion is specific and in malignant cells it results from an autocrine loop. HGF/SF was the only stimulus able to trigger adhesion of HTU-5 cells (hatched bars) to VN. Treatment of HTU-34 cells (open bars) with a blocking polyclonal antiserum to HGF/SF (anti-HGF) markedly reduced adhesion. HGF/SF-induced adhesion in HTU-5 cells was inhibited by mAb LM609 to the β3 subunit (HGF+LM609) and by treating HTU-34–conditioned medium with the inhibitory antiserum against HGF/SF (Cond + anti-HGF). In contrast, addition of preimmune sheep IgGs to HTU-34 cells (P-I) did not modify their adhesive properties. Similarly, treatment of HTU-5 cells with HTU-34–conditioned medium in the presence of preimmune IgGs (Cond + P-I) had no effect on the ability of conditioned medium to trigger cell adhesion. (C) HGF/SF increases adhesion efficiency of HTU-5 cells on FN via the enhancement of αvβ3 ligand-binding ability (see Results). (D) HGF/SF does not modify the adhesive function of laminin-binding integrins. Attachment of both HTU-5 (hatched bars) and HTU-34 (open bars) cells to laminin was inhibited by the β1 function-blocking mAb AIIB2, but was not modulated by HGF/SF treatment.

Mentions: Indeed, HGF/SF clearly promoted attachment and spreading of HTU-5 cells on VN in a dose-dependent manner (Fig. 6 A). HGF/SF-induced adhesion was specifically inhibited by mAb LM609 against αvβ3 (Fig. 6 B). In agreement with these findings, HTU-34 adhesion on VN was impaired by a functional antibody to HGF/SF (Fig. 6 B). Moreover, this antibody, but not normal sheep serum, blocked the ability of HTU-34–conditioned medium to induce adhesion of HTU-5 cells (Fig. 6 B). The proadhesive effect of HGF/SF was specific insofar that TGF-β1 could not enhance HTU-5 cell adhesion to VN (not shown) nor could EGF, insulin, and insulin-like growth factor-1 (Fig. 6 B). It has already been demonstrated that receptors for EGF, insulin, and insulin-like growth factor-1 are present in thyroid cells (Dumont et al., 1991); however, to ascertain that also HTU-5 cells express these receptors, we performed Western blot experiments on total cell lysates after GF stimulation and verified the induction of multiple tyrosine phosphorylated bands (not shown).


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)

HGF/SF selectively  activates the adhesive potential of αvβ3 integrin in  HTU-5 cells. (A) HGF/SF  promotes αvβ3-mediated  adhesion to VN in a dose-dependent manner. Cells  from HTU-5 cultures were  detached, resuspended in  SFM containing increasing  amounts of HGF/SF, and  then plated onto VN. (B)  The effect of HGF/SF on  αvβ3-mediated adhesion is  specific and in malignant  cells it results from an autocrine loop. HGF/SF was the  only stimulus able to trigger  adhesion of HTU-5 cells  (hatched bars) to VN. Treatment of HTU-34 cells (open  bars) with a blocking polyclonal antiserum to HGF/SF  (anti-HGF) markedly reduced adhesion. HGF/SF-induced adhesion in HTU-5  cells was inhibited by mAb  LM609 to the β3 subunit  (HGF+LM609) and by treating HTU-34–conditioned medium with the inhibitory antiserum against HGF/SF (Cond + anti-HGF). In  contrast, addition of preimmune sheep IgGs to HTU-34 cells (P-I) did not modify their adhesive properties. Similarly, treatment of  HTU-5 cells with HTU-34–conditioned medium in the presence of preimmune IgGs (Cond + P-I) had no effect on the ability of conditioned medium to trigger cell adhesion. (C) HGF/SF increases adhesion efficiency of HTU-5 cells on FN via the enhancement of αvβ3  ligand-binding ability (see Results). (D) HGF/SF does not modify the adhesive function of laminin-binding integrins. Attachment of  both HTU-5 (hatched bars) and HTU-34 (open bars) cells to laminin was inhibited by the β1 function-blocking mAb AIIB2, but was not  modulated by HGF/SF treatment.
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Related In: Results  -  Collection

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Figure 6: HGF/SF selectively activates the adhesive potential of αvβ3 integrin in HTU-5 cells. (A) HGF/SF promotes αvβ3-mediated adhesion to VN in a dose-dependent manner. Cells from HTU-5 cultures were detached, resuspended in SFM containing increasing amounts of HGF/SF, and then plated onto VN. (B) The effect of HGF/SF on αvβ3-mediated adhesion is specific and in malignant cells it results from an autocrine loop. HGF/SF was the only stimulus able to trigger adhesion of HTU-5 cells (hatched bars) to VN. Treatment of HTU-34 cells (open bars) with a blocking polyclonal antiserum to HGF/SF (anti-HGF) markedly reduced adhesion. HGF/SF-induced adhesion in HTU-5 cells was inhibited by mAb LM609 to the β3 subunit (HGF+LM609) and by treating HTU-34–conditioned medium with the inhibitory antiserum against HGF/SF (Cond + anti-HGF). In contrast, addition of preimmune sheep IgGs to HTU-34 cells (P-I) did not modify their adhesive properties. Similarly, treatment of HTU-5 cells with HTU-34–conditioned medium in the presence of preimmune IgGs (Cond + P-I) had no effect on the ability of conditioned medium to trigger cell adhesion. (C) HGF/SF increases adhesion efficiency of HTU-5 cells on FN via the enhancement of αvβ3 ligand-binding ability (see Results). (D) HGF/SF does not modify the adhesive function of laminin-binding integrins. Attachment of both HTU-5 (hatched bars) and HTU-34 (open bars) cells to laminin was inhibited by the β1 function-blocking mAb AIIB2, but was not modulated by HGF/SF treatment.
Mentions: Indeed, HGF/SF clearly promoted attachment and spreading of HTU-5 cells on VN in a dose-dependent manner (Fig. 6 A). HGF/SF-induced adhesion was specifically inhibited by mAb LM609 against αvβ3 (Fig. 6 B). In agreement with these findings, HTU-34 adhesion on VN was impaired by a functional antibody to HGF/SF (Fig. 6 B). Moreover, this antibody, but not normal sheep serum, blocked the ability of HTU-34–conditioned medium to induce adhesion of HTU-5 cells (Fig. 6 B). The proadhesive effect of HGF/SF was specific insofar that TGF-β1 could not enhance HTU-5 cell adhesion to VN (not shown) nor could EGF, insulin, and insulin-like growth factor-1 (Fig. 6 B). It has already been demonstrated that receptors for EGF, insulin, and insulin-like growth factor-1 are present in thyroid cells (Dumont et al., 1991); however, to ascertain that also HTU-5 cells express these receptors, we performed Western blot experiments on total cell lysates after GF stimulation and verified the induction of multiple tyrosine phosphorylated bands (not shown).

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