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The role of TG2 in regulating S100A4-mediated mammary tumour cell migration.

Wang Z, Griffin M - PLoS ONE (2013)

Bottom Line: Inhibition was paralleled by a decrease in S100A4 polymer formation.Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and α5β1 integrin co-signalling pathways linked by activation of PKCα in this TG2 and S100A4-mediated cell migration.We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

View Article: PubMed Central - PubMed

Affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, United Kingdom.

ABSTRACT
The importance of S100A4, a Ca(2+)-binding protein, in mediating tumour cell migration, both intracellularly and extracellularly, is well documented. Tissue transglutaminase (TG2) a Ca(2+)-dependent protein crosslinking enzyme, has also been shown to enhance cell migration. Here by using the well characterised non-metastatic rat mammary R37 cells (transfected with empty vector) and highly metastatic KP1 cells (R37 cells transfected with S100A4), we demonstrate that inhibition of TG2 either by TG2 inhibitors or transfection of cells with TG2 shRNA block S100A4-accelerated cell migration in the KP1cells and in R37 cells treated with exogenous S100A4. Cell migration was also blocked by the treatment with the non-cell permeabilizing TG2 inhibitor R294, in the human breast cancer cell line MDA-MB-231 (Clone 16, which has a high level of TG2 expression). Inhibition was paralleled by a decrease in S100A4 polymer formation. In vitro co-immunoprecipitation and Far Western blotting assays and cross-linking assays showed not only the direct interaction between TG2 and S100A4, but also confirmed S100A4 as a substrate for TG2. Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and α5β1 integrin co-signalling pathways linked by activation of PKCα in this TG2 and S100A4-mediated cell migration. We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

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The role S100A4 in TG2-related breast cancer cell migration.(A) The presence of TG2 in MDA-MB-231 cell lines. Western blotting was performed to detect TG2 in MDA-MB-231 wild type and clone 16 cells. (B) Wound healing assays for clone 16 and wild type MDA-MB-231 cells with or without treatment with TG2 inhibitor R294. (C) The importance of TG2 and S100A4 in MDA-MB-231 breast cancer cell migration. MDA-MB-231 wild type and clone 16 cells were used in the wound healing assay in the presence of TG2 antibodies (Cub7402, TG100 and 1D10) and anti-S100A4 antibodies (rabbit polyclonal and mouse monoclonal) at the concentrations of 20 μg/ml, while TG2 inhibitor R294 was used at the concentration of 0.25 mM. Mouse control IgG and rabbit control IgG were used as the control treatments. At least 3 images were taken from each wound to analyse the closure rate of the wound and three separate experiments were performed. (D) S100A4 in MDA-MB-231 clone 16 cells. Western blotting was performed to detect the presence of S100A4 antigen in clone 16 cells in the presence or absence of TG2 inhibitor R294 by using specific anti-S100A4 antibody.
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pone-0057017-g011: The role S100A4 in TG2-related breast cancer cell migration.(A) The presence of TG2 in MDA-MB-231 cell lines. Western blotting was performed to detect TG2 in MDA-MB-231 wild type and clone 16 cells. (B) Wound healing assays for clone 16 and wild type MDA-MB-231 cells with or without treatment with TG2 inhibitor R294. (C) The importance of TG2 and S100A4 in MDA-MB-231 breast cancer cell migration. MDA-MB-231 wild type and clone 16 cells were used in the wound healing assay in the presence of TG2 antibodies (Cub7402, TG100 and 1D10) and anti-S100A4 antibodies (rabbit polyclonal and mouse monoclonal) at the concentrations of 20 μg/ml, while TG2 inhibitor R294 was used at the concentration of 0.25 mM. Mouse control IgG and rabbit control IgG were used as the control treatments. At least 3 images were taken from each wound to analyse the closure rate of the wound and three separate experiments were performed. (D) S100A4 in MDA-MB-231 clone 16 cells. Western blotting was performed to detect the presence of S100A4 antigen in clone 16 cells in the presence or absence of TG2 inhibitor R294 by using specific anti-S100A4 antibody.

Mentions: The human breast cancer cell line MDA-MB-231 was used to extend our observation for the importance of both TG2 and S100A4 proteins in R37 and KP1 cells to human cancer cells. Higher TG2 expression was detected in the clone 16 cells, while negligible TG2 was found in the wild type MDA-MB-231 cells (Fig. 11A). In the wound healing assay, increased migration was observed in the high TG2 expressing clone16 cells when compared to the wild type MDA-MB-231 cells which contain very low levels of TG2 (Fig. 11B). Importantly in addition to R294 the TG2 monoclonal antibodies Cub7402, TG100 and 1D10 and the polyclonal antibody for S100A4 could block the migration of these cells. As found with KP1 cells, this suggests the involvement of extracellular TG2 and S100A4 in regulating MDA-MB-231 clone 16 cell migration (Fig. 11C). Western blotting for S100A4 antigen in clone16 cells showed the presence of high-molecular weight S100A4 polymers, comparable to those found in the in vitro crosslinking assay and in the KP1 cells, which could be reduced by treatment of cells with the non-cell permeable TG2 inhibitor R294 (Fig. 11D).


The role of TG2 in regulating S100A4-mediated mammary tumour cell migration.

Wang Z, Griffin M - PLoS ONE (2013)

The role S100A4 in TG2-related breast cancer cell migration.(A) The presence of TG2 in MDA-MB-231 cell lines. Western blotting was performed to detect TG2 in MDA-MB-231 wild type and clone 16 cells. (B) Wound healing assays for clone 16 and wild type MDA-MB-231 cells with or without treatment with TG2 inhibitor R294. (C) The importance of TG2 and S100A4 in MDA-MB-231 breast cancer cell migration. MDA-MB-231 wild type and clone 16 cells were used in the wound healing assay in the presence of TG2 antibodies (Cub7402, TG100 and 1D10) and anti-S100A4 antibodies (rabbit polyclonal and mouse monoclonal) at the concentrations of 20 μg/ml, while TG2 inhibitor R294 was used at the concentration of 0.25 mM. Mouse control IgG and rabbit control IgG were used as the control treatments. At least 3 images were taken from each wound to analyse the closure rate of the wound and three separate experiments were performed. (D) S100A4 in MDA-MB-231 clone 16 cells. Western blotting was performed to detect the presence of S100A4 antigen in clone 16 cells in the presence or absence of TG2 inhibitor R294 by using specific anti-S100A4 antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585722&req=5

pone-0057017-g011: The role S100A4 in TG2-related breast cancer cell migration.(A) The presence of TG2 in MDA-MB-231 cell lines. Western blotting was performed to detect TG2 in MDA-MB-231 wild type and clone 16 cells. (B) Wound healing assays for clone 16 and wild type MDA-MB-231 cells with or without treatment with TG2 inhibitor R294. (C) The importance of TG2 and S100A4 in MDA-MB-231 breast cancer cell migration. MDA-MB-231 wild type and clone 16 cells were used in the wound healing assay in the presence of TG2 antibodies (Cub7402, TG100 and 1D10) and anti-S100A4 antibodies (rabbit polyclonal and mouse monoclonal) at the concentrations of 20 μg/ml, while TG2 inhibitor R294 was used at the concentration of 0.25 mM. Mouse control IgG and rabbit control IgG were used as the control treatments. At least 3 images were taken from each wound to analyse the closure rate of the wound and three separate experiments were performed. (D) S100A4 in MDA-MB-231 clone 16 cells. Western blotting was performed to detect the presence of S100A4 antigen in clone 16 cells in the presence or absence of TG2 inhibitor R294 by using specific anti-S100A4 antibody.
Mentions: The human breast cancer cell line MDA-MB-231 was used to extend our observation for the importance of both TG2 and S100A4 proteins in R37 and KP1 cells to human cancer cells. Higher TG2 expression was detected in the clone 16 cells, while negligible TG2 was found in the wild type MDA-MB-231 cells (Fig. 11A). In the wound healing assay, increased migration was observed in the high TG2 expressing clone16 cells when compared to the wild type MDA-MB-231 cells which contain very low levels of TG2 (Fig. 11B). Importantly in addition to R294 the TG2 monoclonal antibodies Cub7402, TG100 and 1D10 and the polyclonal antibody for S100A4 could block the migration of these cells. As found with KP1 cells, this suggests the involvement of extracellular TG2 and S100A4 in regulating MDA-MB-231 clone 16 cell migration (Fig. 11C). Western blotting for S100A4 antigen in clone16 cells showed the presence of high-molecular weight S100A4 polymers, comparable to those found in the in vitro crosslinking assay and in the KP1 cells, which could be reduced by treatment of cells with the non-cell permeable TG2 inhibitor R294 (Fig. 11D).

Bottom Line: Inhibition was paralleled by a decrease in S100A4 polymer formation.Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and α5β1 integrin co-signalling pathways linked by activation of PKCα in this TG2 and S100A4-mediated cell migration.We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

View Article: PubMed Central - PubMed

Affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, United Kingdom.

ABSTRACT
The importance of S100A4, a Ca(2+)-binding protein, in mediating tumour cell migration, both intracellularly and extracellularly, is well documented. Tissue transglutaminase (TG2) a Ca(2+)-dependent protein crosslinking enzyme, has also been shown to enhance cell migration. Here by using the well characterised non-metastatic rat mammary R37 cells (transfected with empty vector) and highly metastatic KP1 cells (R37 cells transfected with S100A4), we demonstrate that inhibition of TG2 either by TG2 inhibitors or transfection of cells with TG2 shRNA block S100A4-accelerated cell migration in the KP1cells and in R37 cells treated with exogenous S100A4. Cell migration was also blocked by the treatment with the non-cell permeabilizing TG2 inhibitor R294, in the human breast cancer cell line MDA-MB-231 (Clone 16, which has a high level of TG2 expression). Inhibition was paralleled by a decrease in S100A4 polymer formation. In vitro co-immunoprecipitation and Far Western blotting assays and cross-linking assays showed not only the direct interaction between TG2 and S100A4, but also confirmed S100A4 as a substrate for TG2. Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and α5β1 integrin co-signalling pathways linked by activation of PKCα in this TG2 and S100A4-mediated cell migration. We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

Show MeSH
Related in: MedlinePlus