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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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Identification of TG2 in R37 and KP1 cells.(A) The presence of TG2 and TG1 in R37 and KP1 cells. Whole cell lysates from R37 and KP1 cells were prepared as described in the Materials and Methods. Western blotting was used to detect the presence of TG2 and TG1 antigen by using the specific monoclonal TG2 antibody Cub7402 and a polyclonal anti-TG2 antibody and a polyclonal antibody against TG1, while α-Tubulin was used as the equal loading standard for densitometric analysis, as shown in the inset histogram (mean ± S.D. from two separate experiments undertaken in duplicate). (B) Detection of TG2 activity in R37 and KP1 cell lysates. A TG2-specific CovTest assay was performed using R37 and KP1 cell lysates (50 µg protein) as described in the Material and Methods. Data represent mean values ± S.D. from 3 separate experiments. (C) Cell surface TG activity was undertaken with live cells (2×104 cells/well). Cells were incubated with biotin-cadaverine in serum free medium for 2 h after plating onto FN coated wells. HRP conjugated Extr-avidin was used to detect the biotin-labelled crosslinking products as described in the Materials and Methods. Data represent mean values ± S.D. from 3 separate experiments. *, p<0.05 compared to the control sample.
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pone-0057017-g004: Identification of TG2 in R37 and KP1 cells.(A) The presence of TG2 and TG1 in R37 and KP1 cells. Whole cell lysates from R37 and KP1 cells were prepared as described in the Materials and Methods. Western blotting was used to detect the presence of TG2 and TG1 antigen by using the specific monoclonal TG2 antibody Cub7402 and a polyclonal anti-TG2 antibody and a polyclonal antibody against TG1, while α-Tubulin was used as the equal loading standard for densitometric analysis, as shown in the inset histogram (mean ± S.D. from two separate experiments undertaken in duplicate). (B) Detection of TG2 activity in R37 and KP1 cell lysates. A TG2-specific CovTest assay was performed using R37 and KP1 cell lysates (50 µg protein) as described in the Material and Methods. Data represent mean values ± S.D. from 3 separate experiments. (C) Cell surface TG activity was undertaken with live cells (2×104 cells/well). Cells were incubated with biotin-cadaverine in serum free medium for 2 h after plating onto FN coated wells. HRP conjugated Extr-avidin was used to detect the biotin-labelled crosslinking products as described in the Materials and Methods. Data represent mean values ± S.D. from 3 separate experiments. *, p<0.05 compared to the control sample.

Mentions: To confirm the presence of TG2 in R37 and KP1 cells, Western blotting was performed to detect the antigen in these cells by using both mouse monoclonal anti-TG2 antibody Cub7402 and a rabbit polyclonal anti-TG2 antibody. Easily detectable amounts of TG2 antigen were present in the cell lysates of both R37 and KP1 cells, although higher (approx. 20%) in the R37 cells (Fig. 4A). Also small amounts of the other TG family member, TG1 normally found in cultured epithelial cells could be detected via Western blotting in both cell lines (Fig. 4A). The TG2-specific commercial assay for TG2 activity (TG2-CovTest assay) demonstrated activity in the cell lysates of both cells, although as found with Western blots slightly greater in the R37 cells (Fig. 4B). Measurement of cell surface activity by biotin-cadaverine incorporation into FN confirmed the presence of cell surface TG2 activity in both cells, but unlike cell lysate activity was found to be significantly greater in the KP1 cells (Fig. 4C). Importantly, the TG specific inhibitor R294 (which does not inhibit extracellular Factor XIIIa at the concentration of 0.25 mM) and the TG2 specific inhibitor Z-DON [27]–[29] reduced the cell surface TG activity in both cell lines, suggesting that TG2 is the major cell surface source of the TG activity.


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

Wang Z, Griffin M - PLoS ONE (2013)

Identification of TG2 in R37 and KP1 cells.(A) The presence of TG2 and TG1 in R37 and KP1 cells. Whole cell lysates from R37 and KP1 cells were prepared as described in the Materials and Methods. Western blotting was used to detect the presence of TG2 and TG1 antigen by using the specific monoclonal TG2 antibody Cub7402 and a polyclonal anti-TG2 antibody and a polyclonal antibody against TG1, while α-Tubulin was used as the equal loading standard for densitometric analysis, as shown in the inset histogram (mean ± S.D. from two separate experiments undertaken in duplicate). (B) Detection of TG2 activity in R37 and KP1 cell lysates. A TG2-specific CovTest assay was performed using R37 and KP1 cell lysates (50 µg protein) as described in the Material and Methods. Data represent mean values ± S.D. from 3 separate experiments. (C) Cell surface TG activity was undertaken with live cells (2×104 cells/well). Cells were incubated with biotin-cadaverine in serum free medium for 2 h after plating onto FN coated wells. HRP conjugated Extr-avidin was used to detect the biotin-labelled crosslinking products as described in the Materials and Methods. Data represent mean values ± S.D. from 3 separate experiments. *, p<0.05 compared to the control sample.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585722&req=5

pone-0057017-g004: Identification of TG2 in R37 and KP1 cells.(A) The presence of TG2 and TG1 in R37 and KP1 cells. Whole cell lysates from R37 and KP1 cells were prepared as described in the Materials and Methods. Western blotting was used to detect the presence of TG2 and TG1 antigen by using the specific monoclonal TG2 antibody Cub7402 and a polyclonal anti-TG2 antibody and a polyclonal antibody against TG1, while α-Tubulin was used as the equal loading standard for densitometric analysis, as shown in the inset histogram (mean ± S.D. from two separate experiments undertaken in duplicate). (B) Detection of TG2 activity in R37 and KP1 cell lysates. A TG2-specific CovTest assay was performed using R37 and KP1 cell lysates (50 µg protein) as described in the Material and Methods. Data represent mean values ± S.D. from 3 separate experiments. (C) Cell surface TG activity was undertaken with live cells (2×104 cells/well). Cells were incubated with biotin-cadaverine in serum free medium for 2 h after plating onto FN coated wells. HRP conjugated Extr-avidin was used to detect the biotin-labelled crosslinking products as described in the Materials and Methods. Data represent mean values ± S.D. from 3 separate experiments. *, p<0.05 compared to the control sample.
Mentions: To confirm the presence of TG2 in R37 and KP1 cells, Western blotting was performed to detect the antigen in these cells by using both mouse monoclonal anti-TG2 antibody Cub7402 and a rabbit polyclonal anti-TG2 antibody. Easily detectable amounts of TG2 antigen were present in the cell lysates of both R37 and KP1 cells, although higher (approx. 20%) in the R37 cells (Fig. 4A). Also small amounts of the other TG family member, TG1 normally found in cultured epithelial cells could be detected via Western blotting in both cell lines (Fig. 4A). The TG2-specific commercial assay for TG2 activity (TG2-CovTest assay) demonstrated activity in the cell lysates of both cells, although as found with Western blots slightly greater in the R37 cells (Fig. 4B). Measurement of cell surface activity by biotin-cadaverine incorporation into FN confirmed the presence of cell surface TG2 activity in both cells, but unlike cell lysate activity was found to be significantly greater in the KP1 cells (Fig. 4C). Importantly, the TG specific inhibitor R294 (which does not inhibit extracellular Factor XIIIa at the concentration of 0.25 mM) and the TG2 specific inhibitor Z-DON [27]–[29] reduced the cell surface TG activity in both cell lines, suggesting that TG2 is the major cell surface source of the TG activity.

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