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TGFbeta1 signaling via alphaVbeta6 integrin.

Kracklauer MP, Schmidt C, Sclabas GM - Mol. Cancer (2003)

Bottom Line: Our data provide support for the existence of an alternate TGFbeta1 signaling pathway that is independent of the known SMAD pathway.This alternate pathway involves alphaVbeta6 integrin and the Ras/MAP kinase pathway and does not employ an RGD motif in TGFbeta1-sensitive tumor cells.The combined action of these two pathways seems to be necessary to elicit a complete TGFbeta1 signal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station, A4800, 78712, Austin, TX, USA. mordechai30@hotmail.com

ABSTRACT

Background: Transforming growth factor beta1 (TGFbeta1) is a potent inhibitor of epithelial cell growth, thus playing an important role in tissue homeostasis. Most carcinoma cells exhibit a reduced sensitivity for TGFbeta1 mediated growth inhibition, suggesting TGFbeta1 participation in the development of these cancers. The tumor suppressor gene DPC4/SMAD4, which is frequently inactivated in carcinoma cells, has been described as a key player in TGFbeta1 mediated growth inhibition. However, some carcinoma cells lacking functional SMAD4 are sensitive to TGFbeta1 induced growth inhibition, thus requiring a SMAD4 independent TGFbeta1 pathway.

Results: Here we report that mature TGFbeta1 is a ligand for the integrin alphaVbeta6, independent of the common integrin binding sequence motif RGD. After TGFbeta1 binds to alphaVbeta6 integrin, different signaling proteins are activated in TGFbeta1-sensitive carcinoma cells, but not in cells that are insensitive to TGFbeta1. Among others, interaction of TGFbeta1 with the alphaVbeta6 integrin resulted in an upregulation of the cell cycle inhibitors p21/WAF1 and p27 leading to growth inhibition in SMAD4 deleted as well as in SMAD4 wildtype carcinoma cells.

Conclusions: Our data provide support for the existence of an alternate TGFbeta1 signaling pathway that is independent of the known SMAD pathway. This alternate pathway involves alphaVbeta6 integrin and the Ras/MAP kinase pathway and does not employ an RGD motif in TGFbeta1-sensitive tumor cells. The combined action of these two pathways seems to be necessary to elicit a complete TGFbeta1 signal.

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Enhanced Tyrosine Phosphorylation of proteins associated with the integrin-cytoskeleton-complex. Cytoskeletally anchored αVβ6 was immunoprecipitated after TGFβ1 and/or fibronectin stimulation (10 nM for 10 minutes) followed by Western analysis with antibodies against tyrosine-phosphorylated proteins (A). Reprobing with αV and β6 antibodies show equal anounts of precipitates used (B).
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Figure 3: Enhanced Tyrosine Phosphorylation of proteins associated with the integrin-cytoskeleton-complex. Cytoskeletally anchored αVβ6 was immunoprecipitated after TGFβ1 and/or fibronectin stimulation (10 nM for 10 minutes) followed by Western analysis with antibodies against tyrosine-phosphorylated proteins (A). Reprobing with αV and β6 antibodies show equal anounts of precipitates used (B).

Mentions: To determine whether binding of mature TGFβ1 leads to integrin-mediated signaling, we looked at the status of integrin-cytoskeleton-associated proteins [66,67] after incubation with mature TGFβ1 in selected carcinoma cell lines with different degrees of sensitivity to TGFβ1 (Table 1). Cytoskeletal anchored proteins were precipitated with anti αV and β6-antibodies. Immobilization of proteins to the cytoskeleton (Triton-X insoluble fraction, Figure 2B) as well as tyrosine phosphorylation of these proteins (Figure 2A) induced through mature TGFβ1 was only seen in the TGFβ1-sensitive carcinoma cell lines (Figure 2 and additional file 5). Notably, tyrosine phosphorylation of cytoskeletally anchored proteins is further enhanced after combined treatment with mature TGFβ1 and fibronectin in TGFβ1 sensitive cells (Figure 3). In contrast, in the TGFβ1-resistant AsPC-1 and Capan-1 cells, the interaction of mature TGFβ1 with αVβ6 integrin resulted in an immobilization of high molecular weight proteins to the cytoskeleton without tyrosine phosphorylation (Figure 2). Again, stimulation of TGFβ1 sensitive cells BxPC-3, LoVo [68], SW48 [68], Keratinocytes, HeLa and DLD1 [69], results in an enhanced cytoskeletal immobilization and tyrosine phosphorylation of cellular proteins in response to stimulation with mature TGFβ1 (additional file 5). Remarkably, preincubation with the MEK1 inhibitor PD98059 resulted in a reduced cytoskeletal immobilization and tyrosine phosphorylation of cellular proteins in response to stimulation with mature TGFβ1. This finding is in agreement with other observations that MEK1-mediated signal transduction is involved in cytoskeletal remodeling and integrin engagement [70,71].


TGFbeta1 signaling via alphaVbeta6 integrin.

Kracklauer MP, Schmidt C, Sclabas GM - Mol. Cancer (2003)

Enhanced Tyrosine Phosphorylation of proteins associated with the integrin-cytoskeleton-complex. Cytoskeletally anchored αVβ6 was immunoprecipitated after TGFβ1 and/or fibronectin stimulation (10 nM for 10 minutes) followed by Western analysis with antibodies against tyrosine-phosphorylated proteins (A). Reprobing with αV and β6 antibodies show equal anounts of precipitates used (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC184456&req=5

Figure 3: Enhanced Tyrosine Phosphorylation of proteins associated with the integrin-cytoskeleton-complex. Cytoskeletally anchored αVβ6 was immunoprecipitated after TGFβ1 and/or fibronectin stimulation (10 nM for 10 minutes) followed by Western analysis with antibodies against tyrosine-phosphorylated proteins (A). Reprobing with αV and β6 antibodies show equal anounts of precipitates used (B).
Mentions: To determine whether binding of mature TGFβ1 leads to integrin-mediated signaling, we looked at the status of integrin-cytoskeleton-associated proteins [66,67] after incubation with mature TGFβ1 in selected carcinoma cell lines with different degrees of sensitivity to TGFβ1 (Table 1). Cytoskeletal anchored proteins were precipitated with anti αV and β6-antibodies. Immobilization of proteins to the cytoskeleton (Triton-X insoluble fraction, Figure 2B) as well as tyrosine phosphorylation of these proteins (Figure 2A) induced through mature TGFβ1 was only seen in the TGFβ1-sensitive carcinoma cell lines (Figure 2 and additional file 5). Notably, tyrosine phosphorylation of cytoskeletally anchored proteins is further enhanced after combined treatment with mature TGFβ1 and fibronectin in TGFβ1 sensitive cells (Figure 3). In contrast, in the TGFβ1-resistant AsPC-1 and Capan-1 cells, the interaction of mature TGFβ1 with αVβ6 integrin resulted in an immobilization of high molecular weight proteins to the cytoskeleton without tyrosine phosphorylation (Figure 2). Again, stimulation of TGFβ1 sensitive cells BxPC-3, LoVo [68], SW48 [68], Keratinocytes, HeLa and DLD1 [69], results in an enhanced cytoskeletal immobilization and tyrosine phosphorylation of cellular proteins in response to stimulation with mature TGFβ1 (additional file 5). Remarkably, preincubation with the MEK1 inhibitor PD98059 resulted in a reduced cytoskeletal immobilization and tyrosine phosphorylation of cellular proteins in response to stimulation with mature TGFβ1. This finding is in agreement with other observations that MEK1-mediated signal transduction is involved in cytoskeletal remodeling and integrin engagement [70,71].

Bottom Line: Our data provide support for the existence of an alternate TGFbeta1 signaling pathway that is independent of the known SMAD pathway.This alternate pathway involves alphaVbeta6 integrin and the Ras/MAP kinase pathway and does not employ an RGD motif in TGFbeta1-sensitive tumor cells.The combined action of these two pathways seems to be necessary to elicit a complete TGFbeta1 signal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station, A4800, 78712, Austin, TX, USA. mordechai30@hotmail.com

ABSTRACT

Background: Transforming growth factor beta1 (TGFbeta1) is a potent inhibitor of epithelial cell growth, thus playing an important role in tissue homeostasis. Most carcinoma cells exhibit a reduced sensitivity for TGFbeta1 mediated growth inhibition, suggesting TGFbeta1 participation in the development of these cancers. The tumor suppressor gene DPC4/SMAD4, which is frequently inactivated in carcinoma cells, has been described as a key player in TGFbeta1 mediated growth inhibition. However, some carcinoma cells lacking functional SMAD4 are sensitive to TGFbeta1 induced growth inhibition, thus requiring a SMAD4 independent TGFbeta1 pathway.

Results: Here we report that mature TGFbeta1 is a ligand for the integrin alphaVbeta6, independent of the common integrin binding sequence motif RGD. After TGFbeta1 binds to alphaVbeta6 integrin, different signaling proteins are activated in TGFbeta1-sensitive carcinoma cells, but not in cells that are insensitive to TGFbeta1. Among others, interaction of TGFbeta1 with the alphaVbeta6 integrin resulted in an upregulation of the cell cycle inhibitors p21/WAF1 and p27 leading to growth inhibition in SMAD4 deleted as well as in SMAD4 wildtype carcinoma cells.

Conclusions: Our data provide support for the existence of an alternate TGFbeta1 signaling pathway that is independent of the known SMAD pathway. This alternate pathway involves alphaVbeta6 integrin and the Ras/MAP kinase pathway and does not employ an RGD motif in TGFbeta1-sensitive tumor cells. The combined action of these two pathways seems to be necessary to elicit a complete TGFbeta1 signal.

Show MeSH
Related in: MedlinePlus