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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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Activation and nuclear translocation of SMAD2/3 in response to TGFβ1 (A). Nuclear and cytoplasmatic fraction of cellular proteins (BxPC-3) after stimulation with 10 nM of TGFβ1 for 10 minutes and Western blot analysis for SMAD2/3 and phosphorylated SMAD2/3. Purity of cytoplasmic and nuclear fraction (B). Cytoplasmic and nuclear extracts from K562 cells were probed with p125FAK, PCNA and Iκ Bα antibodies at the same time. As predicted, p125FAK cold exclusively be detected in the cytoplasmic extract, whereas PCNA is found in the nucleus. Iκ Bα served as loading control.
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Figure 9: Activation and nuclear translocation of SMAD2/3 in response to TGFβ1 (A). Nuclear and cytoplasmatic fraction of cellular proteins (BxPC-3) after stimulation with 10 nM of TGFβ1 for 10 minutes and Western blot analysis for SMAD2/3 and phosphorylated SMAD2/3. Purity of cytoplasmic and nuclear fraction (B). Cytoplasmic and nuclear extracts from K562 cells were probed with p125FAK, PCNA and Iκ Bα antibodies at the same time. As predicted, p125FAK cold exclusively be detected in the cytoplasmic extract, whereas PCNA is found in the nucleus. Iκ Bα served as loading control.

Mentions: To explain the TGFβ1 sensitivity of SMAD4-deleted cells, it is speculated that after binding of TGFβ1 to its receptor, activated SMAD2/3 may translocate to the nucleus and activate gene expression even in the absence of SMAD4. To exclude this possibility, cellular proteins were divided into cytoplasmatic and nuclear fractions after TGFβ1 stimulation, and localization and phosphorylation of SMAD2/3 were investigated. In the SMAD4 deleted BxPC-3 cells, TGFβ1 resulted in phosphorylation of SMAD2/3, but the activated SMAD proteins were retained in the cytoplasmatic fraction (Figure 9). Remarkably, in NP-9 cells [74], SMAD2/3 are translocated into the nucleus upon TGFβ1 stimulation (additional file 9(A)) but we could not observe an enhanced tyrosine phosphorylation of cytoskeletal anchored proteins (additional file 9(B)).


TGFbeta1 signaling via alphaVbeta6 integrin.

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

Activation and nuclear translocation of SMAD2/3 in response to TGFβ1 (A). Nuclear and cytoplasmatic fraction of cellular proteins (BxPC-3) after stimulation with 10 nM of TGFβ1 for 10 minutes and Western blot analysis for SMAD2/3 and phosphorylated SMAD2/3. Purity of cytoplasmic and nuclear fraction (B). Cytoplasmic and nuclear extracts from K562 cells were probed with p125FAK, PCNA and Iκ Bα antibodies at the same time. As predicted, p125FAK cold exclusively be detected in the cytoplasmic extract, whereas PCNA is found in the nucleus. Iκ Bα served as loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC184456&req=5

Figure 9: Activation and nuclear translocation of SMAD2/3 in response to TGFβ1 (A). Nuclear and cytoplasmatic fraction of cellular proteins (BxPC-3) after stimulation with 10 nM of TGFβ1 for 10 minutes and Western blot analysis for SMAD2/3 and phosphorylated SMAD2/3. Purity of cytoplasmic and nuclear fraction (B). Cytoplasmic and nuclear extracts from K562 cells were probed with p125FAK, PCNA and Iκ Bα antibodies at the same time. As predicted, p125FAK cold exclusively be detected in the cytoplasmic extract, whereas PCNA is found in the nucleus. Iκ Bα served as loading control.
Mentions: To explain the TGFβ1 sensitivity of SMAD4-deleted cells, it is speculated that after binding of TGFβ1 to its receptor, activated SMAD2/3 may translocate to the nucleus and activate gene expression even in the absence of SMAD4. To exclude this possibility, cellular proteins were divided into cytoplasmatic and nuclear fractions after TGFβ1 stimulation, and localization and phosphorylation of SMAD2/3 were investigated. In the SMAD4 deleted BxPC-3 cells, TGFβ1 resulted in phosphorylation of SMAD2/3, but the activated SMAD proteins were retained in the cytoplasmatic fraction (Figure 9). Remarkably, in NP-9 cells [74], SMAD2/3 are translocated into the nucleus upon TGFβ1 stimulation (additional file 9(A)) but we could not observe an enhanced tyrosine phosphorylation of cytoskeletal anchored proteins (additional file 9(B)).

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