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TGFbeta1 activates c-Jun and Erk1 via alphaVbeta6 integrin.

Luettich K, Schmidt C - Mol. Cancer (2003)

Bottom Line: Overexpression of alphaVbeta6 integrin is associated with lymph node metastasis of gastric carcinomas.We conclude that TGFbeta1 activates c-Jun via the MEKK1/p38 MAP kinase pathway and influences cytoskeletal organization.These finding may provide a link between TGFbeta1 and the metastatic behavior of cancers.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Department of Dermatology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York, 10021, USA. karsta.luettich@molecular-cancer.org

ABSTRACT
Transforming growth factor beta (TGFbeta) plays an important role in animal development and many cellular processes. A variety of cellular functions that are required for tumor metastasis are controlled by integrins, a family of cell adhesion receptors. Overexpression of alphaVbeta6 integrin is associated with lymph node metastasis of gastric carcinomas. It has been demonstrated that a full TGFbeta1 signal requires both alphaVbeta6 integrin and SMAD pathway. TGFbeta1 binds to alphaVbeta6 via the DLXXL motif, a freely accessible amino acid sequence in the mature form of TGFbeta1. Binding of mature TGFbeta1 to alphaVbeta6 leads to immobilization and tyrosine phosphorylation of proteins, which are associated with focal adhesions, a hallmark of integrin-mediated signal transduction. Here, we show that binding of mature TGFbeta1 recruits the mitogen-activated protein kinase kinase kinase 1 (MEKK1), a mediator of c-Jun activation, and the extracellular signaling-regulated kinase-1 (Erk1) to focal adhesions. In addition, the p21-activated kinase 1 (PAK1) is associated with focal adhesions and differentially phosphorylated upon TGFbeta1 stimulation. We conclude that TGFbeta1 activates c-Jun via the MEKK1/p38 MAP kinase pathway and influences cytoskeletal organization. These finding may provide a link between TGFbeta1 and the metastatic behavior of cancers.

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Colocalization of TGFβ1, αVβ6 integrin and the cytoskeleton. 10,000 cells(SW48, DLD1, HeLa, keratinocytes) were cultured on glass coverslips in DMEM supplemented with 17% of heat inactivated fetal bovine serum and stimulated with 10 nM of mature TGFβ1 (from R&D Systems) for ten minutes. After preparation of the cytoskeletal fraction by Triton-X100 extraction [10], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for actin (sc-8432, Santa Cruz), Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αVβ6 integrin (sc-6617 and sc-6632), and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 (sc-146) labeling and viewed using a Zeiss LSM-510 confocal microscope [10]. Magnification 1000 ×.
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Figure 1: Colocalization of TGFβ1, αVβ6 integrin and the cytoskeleton. 10,000 cells(SW48, DLD1, HeLa, keratinocytes) were cultured on glass coverslips in DMEM supplemented with 17% of heat inactivated fetal bovine serum and stimulated with 10 nM of mature TGFβ1 (from R&D Systems) for ten minutes. After preparation of the cytoskeletal fraction by Triton-X100 extraction [10], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for actin (sc-8432, Santa Cruz), Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αVβ6 integrin (sc-6617 and sc-6632), and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 (sc-146) labeling and viewed using a Zeiss LSM-510 confocal microscope [10]. Magnification 1000 ×.

Mentions: We have demonstrated that mature TGFβ1 colocalizes with αVβ6 integrin in Panc-1 cells [10]. To further support our initial findings, we assayed for colocalization between TGFβ1, αVβ6 integrin and the F-actin filaments of the cytoskeleton in SW48, DLD1, HeLa cells as well as keratinocytes. Cells were stimulated with 10 nM of mature TGFβ1. After preparation of the cytoskeletal fraction by Triton-X100 extraction [10,17,18], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αVβ6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [10]. As shown in Figure 1, in TGFβ1, αVβ6 integrin and the F-Actin filaments of the cytoskeleton colocalize after stimulation with mature TGFβ1 in all cell lines used. These results further support our initial findings that mature TGFβ1 is a ligand for αVβ6 integrin.


TGFbeta1 activates c-Jun and Erk1 via alphaVbeta6 integrin.

Luettich K, Schmidt C - Mol. Cancer (2003)

Colocalization of TGFβ1, αVβ6 integrin and the cytoskeleton. 10,000 cells(SW48, DLD1, HeLa, keratinocytes) were cultured on glass coverslips in DMEM supplemented with 17% of heat inactivated fetal bovine serum and stimulated with 10 nM of mature TGFβ1 (from R&D Systems) for ten minutes. After preparation of the cytoskeletal fraction by Triton-X100 extraction [10], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for actin (sc-8432, Santa Cruz), Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αVβ6 integrin (sc-6617 and sc-6632), and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 (sc-146) labeling and viewed using a Zeiss LSM-510 confocal microscope [10]. Magnification 1000 ×.
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Related In: Results  -  Collection

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Figure 1: Colocalization of TGFβ1, αVβ6 integrin and the cytoskeleton. 10,000 cells(SW48, DLD1, HeLa, keratinocytes) were cultured on glass coverslips in DMEM supplemented with 17% of heat inactivated fetal bovine serum and stimulated with 10 nM of mature TGFβ1 (from R&D Systems) for ten minutes. After preparation of the cytoskeletal fraction by Triton-X100 extraction [10], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for actin (sc-8432, Santa Cruz), Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αVβ6 integrin (sc-6617 and sc-6632), and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 (sc-146) labeling and viewed using a Zeiss LSM-510 confocal microscope [10]. Magnification 1000 ×.
Mentions: We have demonstrated that mature TGFβ1 colocalizes with αVβ6 integrin in Panc-1 cells [10]. To further support our initial findings, we assayed for colocalization between TGFβ1, αVβ6 integrin and the F-actin filaments of the cytoskeleton in SW48, DLD1, HeLa cells as well as keratinocytes. Cells were stimulated with 10 nM of mature TGFβ1. After preparation of the cytoskeletal fraction by Triton-X100 extraction [10,17,18], slides were stained using Alexa Fluor 680 goat anti-mouse IgG (Molecular Probes, Eugene, OR) for Actin, Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes) for αVβ6 integrin, and Alexa Fluor 350 donkey anti-rabbit IgG (Molecular Probes) for TGFβ1 labeling [10]. As shown in Figure 1, in TGFβ1, αVβ6 integrin and the F-Actin filaments of the cytoskeleton colocalize after stimulation with mature TGFβ1 in all cell lines used. These results further support our initial findings that mature TGFβ1 is a ligand for αVβ6 integrin.

Bottom Line: Overexpression of alphaVbeta6 integrin is associated with lymph node metastasis of gastric carcinomas.We conclude that TGFbeta1 activates c-Jun via the MEKK1/p38 MAP kinase pathway and influences cytoskeletal organization.These finding may provide a link between TGFbeta1 and the metastatic behavior of cancers.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Department of Dermatology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York, 10021, USA. karsta.luettich@molecular-cancer.org

ABSTRACT
Transforming growth factor beta (TGFbeta) plays an important role in animal development and many cellular processes. A variety of cellular functions that are required for tumor metastasis are controlled by integrins, a family of cell adhesion receptors. Overexpression of alphaVbeta6 integrin is associated with lymph node metastasis of gastric carcinomas. It has been demonstrated that a full TGFbeta1 signal requires both alphaVbeta6 integrin and SMAD pathway. TGFbeta1 binds to alphaVbeta6 via the DLXXL motif, a freely accessible amino acid sequence in the mature form of TGFbeta1. Binding of mature TGFbeta1 to alphaVbeta6 leads to immobilization and tyrosine phosphorylation of proteins, which are associated with focal adhesions, a hallmark of integrin-mediated signal transduction. Here, we show that binding of mature TGFbeta1 recruits the mitogen-activated protein kinase kinase kinase 1 (MEKK1), a mediator of c-Jun activation, and the extracellular signaling-regulated kinase-1 (Erk1) to focal adhesions. In addition, the p21-activated kinase 1 (PAK1) is associated with focal adhesions and differentially phosphorylated upon TGFbeta1 stimulation. We conclude that TGFbeta1 activates c-Jun via the MEKK1/p38 MAP kinase pathway and influences cytoskeletal organization. These finding may provide a link between TGFbeta1 and the metastatic behavior of cancers.

Show MeSH
Related in: MedlinePlus