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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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Exogenous S100A4 enhances cell migration in R37 cells, which can be blocked by TG inhibitors and agents that interfere with syndecan-4 signalling.(A) The wound healing assay in R37 cells was carried out in the presence of extracellular recombinant S100A4 (exo-S100A4,1 μg/ml) with or without TG2 inhibitor R294 (0.25 mM) or with TG2 crosslinked-S100A4 in (exo-X-S100A4) in the presence of R294. The wound assay was also carried out in the presence of heparinase (15 mU/ml), exogenous recombinant human syndecan-4 (1 μg/ml), α5β1 integrin-targetting peptide A5-1 (5 μM) and PKCα inhibitor Go6976 (5 μM) added in the presence of exogenous S100A4 as described in the Materials and Methods. At least 3 images were taken from each wound to verify the closure rate of the wound and three separate experiments were performed. (B) Shows the mean percentage wound closure mean ± S.D. from 3 separate experiments. *, p<0.05 between the groups as indicated in the figure. (C) Knocking down of TG2 in R37 cells. Western blotting was performed to detect TG2 in R37 stably transfected with TG2 shRNA #3 or the scrambled shRNA control. (D) Wound healing assay was carried out on R37 cells transfected with scrambled or TG2 shRNA #3 in the presence of exogenous S100A4 treatment and analysed (E) As described in the Materials and Methods. *, p<0.05 between the groups as indicated in the figure.
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pone-0057017-g009: Exogenous S100A4 enhances cell migration in R37 cells, which can be blocked by TG inhibitors and agents that interfere with syndecan-4 signalling.(A) The wound healing assay in R37 cells was carried out in the presence of extracellular recombinant S100A4 (exo-S100A4,1 μg/ml) with or without TG2 inhibitor R294 (0.25 mM) or with TG2 crosslinked-S100A4 in (exo-X-S100A4) in the presence of R294. The wound assay was also carried out in the presence of heparinase (15 mU/ml), exogenous recombinant human syndecan-4 (1 μg/ml), α5β1 integrin-targetting peptide A5-1 (5 μM) and PKCα inhibitor Go6976 (5 μM) added in the presence of exogenous S100A4 as described in the Materials and Methods. At least 3 images were taken from each wound to verify the closure rate of the wound and three separate experiments were performed. (B) Shows the mean percentage wound closure mean ± S.D. from 3 separate experiments. *, p<0.05 between the groups as indicated in the figure. (C) Knocking down of TG2 in R37 cells. Western blotting was performed to detect TG2 in R37 stably transfected with TG2 shRNA #3 or the scrambled shRNA control. (D) Wound healing assay was carried out on R37 cells transfected with scrambled or TG2 shRNA #3 in the presence of exogenous S100A4 treatment and analysed (E) As described in the Materials and Methods. *, p<0.05 between the groups as indicated in the figure.

Mentions: Given the cell surface co-localization of TG2 activity and S100A4, the ability of the non-cell permeable TG2 inhibitors R281 and R294 to reduce this and in parallel inhibit KP1 cell migration, our results suggest that the major action of TG2 on S100A4 is likely to be through an extracellular mediated signalling pathway involving the cell surface receptor syndecan-4. To test this hypothesis, recombinant S100A4 protein was first used to exogenously treat the control R37 cells. As shown in Fig. 9A and B the addition of exogenous S100A4 promoted the migration of R37 cells. To confirm that TG2 was also involved in this exogenous S100A4 stimulated migration cells were incubated with the TG2 inhibitor R294, which resulted in significant inhibition of the S100A4 stimulated migration (Fig. 9A and B). Importantly S100A4 crosslinked in vitro and then added back to the inhibited R37 cells could partially compensate the effect of TG2 inhibitor R294 (Fig. 9A and B). To further confirm that the effect of the exogenously added S100A4 shares a similar signalling pathway to that of the KP1 cells which over-express endogenous S100A4, wound healing assays were performed. Following cell wounding, heparinase, recombinant syndecan-4, A5-1 inhibiting peptide against α5β1 integrins and PKCα inhibitor Go6976 were added to the scratched cells resulting in significant inhibition of the exogenously stimulated S100A4 migration (Fig. 9A and B). The knock down of TG2 expression by shRNA (#3) in R37 cells (Fig. 9C), which had no effect on the key receptors β1 and α5 integrin or Syndecan-4 when compared to their scrambled shRNA controls (Fig. S1), also led to the failure of the cells to respond to the exogenous S100A4 treatment in the wound healing assay (Fig. 9D and E), confirming the involvement of TG2 in extracellular S100A4-induced cell migration.


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

Wang Z, Griffin M - PLoS ONE (2013)

Exogenous S100A4 enhances cell migration in R37 cells, which can be blocked by TG inhibitors and agents that interfere with syndecan-4 signalling.(A) The wound healing assay in R37 cells was carried out in the presence of extracellular recombinant S100A4 (exo-S100A4,1 μg/ml) with or without TG2 inhibitor R294 (0.25 mM) or with TG2 crosslinked-S100A4 in (exo-X-S100A4) in the presence of R294. The wound assay was also carried out in the presence of heparinase (15 mU/ml), exogenous recombinant human syndecan-4 (1 μg/ml), α5β1 integrin-targetting peptide A5-1 (5 μM) and PKCα inhibitor Go6976 (5 μM) added in the presence of exogenous S100A4 as described in the Materials and Methods. At least 3 images were taken from each wound to verify the closure rate of the wound and three separate experiments were performed. (B) Shows the mean percentage wound closure mean ± S.D. from 3 separate experiments. *, p<0.05 between the groups as indicated in the figure. (C) Knocking down of TG2 in R37 cells. Western blotting was performed to detect TG2 in R37 stably transfected with TG2 shRNA #3 or the scrambled shRNA control. (D) Wound healing assay was carried out on R37 cells transfected with scrambled or TG2 shRNA #3 in the presence of exogenous S100A4 treatment and analysed (E) As described in the Materials and Methods. *, p<0.05 between the groups as indicated in the figure.
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getmorefigures.php?uid=PMC3585722&req=5

pone-0057017-g009: Exogenous S100A4 enhances cell migration in R37 cells, which can be blocked by TG inhibitors and agents that interfere with syndecan-4 signalling.(A) The wound healing assay in R37 cells was carried out in the presence of extracellular recombinant S100A4 (exo-S100A4,1 μg/ml) with or without TG2 inhibitor R294 (0.25 mM) or with TG2 crosslinked-S100A4 in (exo-X-S100A4) in the presence of R294. The wound assay was also carried out in the presence of heparinase (15 mU/ml), exogenous recombinant human syndecan-4 (1 μg/ml), α5β1 integrin-targetting peptide A5-1 (5 μM) and PKCα inhibitor Go6976 (5 μM) added in the presence of exogenous S100A4 as described in the Materials and Methods. At least 3 images were taken from each wound to verify the closure rate of the wound and three separate experiments were performed. (B) Shows the mean percentage wound closure mean ± S.D. from 3 separate experiments. *, p<0.05 between the groups as indicated in the figure. (C) Knocking down of TG2 in R37 cells. Western blotting was performed to detect TG2 in R37 stably transfected with TG2 shRNA #3 or the scrambled shRNA control. (D) Wound healing assay was carried out on R37 cells transfected with scrambled or TG2 shRNA #3 in the presence of exogenous S100A4 treatment and analysed (E) As described in the Materials and Methods. *, p<0.05 between the groups as indicated in the figure.
Mentions: Given the cell surface co-localization of TG2 activity and S100A4, the ability of the non-cell permeable TG2 inhibitors R281 and R294 to reduce this and in parallel inhibit KP1 cell migration, our results suggest that the major action of TG2 on S100A4 is likely to be through an extracellular mediated signalling pathway involving the cell surface receptor syndecan-4. To test this hypothesis, recombinant S100A4 protein was first used to exogenously treat the control R37 cells. As shown in Fig. 9A and B the addition of exogenous S100A4 promoted the migration of R37 cells. To confirm that TG2 was also involved in this exogenous S100A4 stimulated migration cells were incubated with the TG2 inhibitor R294, which resulted in significant inhibition of the S100A4 stimulated migration (Fig. 9A and B). Importantly S100A4 crosslinked in vitro and then added back to the inhibited R37 cells could partially compensate the effect of TG2 inhibitor R294 (Fig. 9A and B). To further confirm that the effect of the exogenously added S100A4 shares a similar signalling pathway to that of the KP1 cells which over-express endogenous S100A4, wound healing assays were performed. Following cell wounding, heparinase, recombinant syndecan-4, A5-1 inhibiting peptide against α5β1 integrins and PKCα inhibitor Go6976 were added to the scratched cells resulting in significant inhibition of the exogenously stimulated S100A4 migration (Fig. 9A and B). The knock down of TG2 expression by shRNA (#3) in R37 cells (Fig. 9C), which had no effect on the key receptors β1 and α5 integrin or Syndecan-4 when compared to their scrambled shRNA controls (Fig. S1), also led to the failure of the cells to respond to the exogenous S100A4 treatment in the wound healing assay (Fig. 9D and E), confirming the involvement of TG2 in extracellular S100A4-induced cell migration.

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