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Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma.

Caffarel MM, Chattopadhyay A, Araujo AM, Bauer J, Scarpini CG, Coleman N - J. Pathol. (2013)

Bottom Line: Cervical SCC cells that over-express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro-malignant effects, including increased cell migration and invasiveness.Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in SCC cells.We conclude that an OSMR/TGM2/integrin-α5β1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, UK.

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Related in: MedlinePlus

Interactions between TGM2 and integrin–α5β1 in cervical SCC cells. (A) Flow-cytometric quantification of cell-surface TGM2 expressed by SW756 and CaSki, comparing cells treated with OSM for 48 h (OSM) with control cells treated with vehicle only (C). Negative control cells were stained with an isotype control IgG. (B) Immunofluorescence for TGM2 (green) and integrin–α5 (red) in SW756 cells treated with vehicle (control) or OSM (48 h) for 48 h, showing focal co-localization (yellow); scale bars = 10 µm. (C) Immunoprecipitation showing physical interaction between TGM2 and integrin–α5β1 in SW756 and CaSki. Cells cultured on fibronectin-coated plates and treated with vehicle (C) or OSM (OSM) for 48 h were immunoprecipitated with anti-integrin–α5β1 antibodies or with IgG isotype-matched control antibodies. The immunoprecipitated (IP) and unbound (UNB) fractions, together with whole-cell lysates, were then analysed by western blotting, using antibodies against TGM2 (top row), integrin–α5 (ITGA5; middle row) and integrin–β1 (ITGB1; bottom row). The anti-integrin–α5β1 antibody precipitated TGM2 in both cell lines (cf lanes 3 and 4 versus 7 and 8 for each panel), with a stronger band for SW756 than for CaSki. Increased levels of precipitated protein were seen in OSM-treated cells (cf lanes 4 versus 3 in each panel). (D) Western blot showing levels of Y397-phosphorylated Fak (P-Fak) and total Fak in SW756 and CaSki cells cultured on fibronectin-coated plates and treated with OSM or vehicle (C) for 48 h
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fig04: Interactions between TGM2 and integrin–α5β1 in cervical SCC cells. (A) Flow-cytometric quantification of cell-surface TGM2 expressed by SW756 and CaSki, comparing cells treated with OSM for 48 h (OSM) with control cells treated with vehicle only (C). Negative control cells were stained with an isotype control IgG. (B) Immunofluorescence for TGM2 (green) and integrin–α5 (red) in SW756 cells treated with vehicle (control) or OSM (48 h) for 48 h, showing focal co-localization (yellow); scale bars = 10 µm. (C) Immunoprecipitation showing physical interaction between TGM2 and integrin–α5β1 in SW756 and CaSki. Cells cultured on fibronectin-coated plates and treated with vehicle (C) or OSM (OSM) for 48 h were immunoprecipitated with anti-integrin–α5β1 antibodies or with IgG isotype-matched control antibodies. The immunoprecipitated (IP) and unbound (UNB) fractions, together with whole-cell lysates, were then analysed by western blotting, using antibodies against TGM2 (top row), integrin–α5 (ITGA5; middle row) and integrin–β1 (ITGB1; bottom row). The anti-integrin–α5β1 antibody precipitated TGM2 in both cell lines (cf lanes 3 and 4 versus 7 and 8 for each panel), with a stronger band for SW756 than for CaSki. Increased levels of precipitated protein were seen in OSM-treated cells (cf lanes 4 versus 3 in each panel). (D) Western blot showing levels of Y397-phosphorylated Fak (P-Fak) and total Fak in SW756 and CaSki cells cultured on fibronectin-coated plates and treated with OSM or vehicle (C) for 48 h

Mentions: By flow cytometry, we observed that OSM treatment significantly increased expression of integrin–α5β1 dimers at the cell surface in both SW756 and CaSki (Figure 3B). The function of TGM2 relates to its subcellular location, with cell surface protein being responsible for interactions with integrins 8. Flow cytometry demonstrated the presence of TGM2 on the cell surface of both SW756 and CaSki (Figure 4A). OSM treatment increased the levels of membrane-associated TGM2, although the differences did not reach statistical significance. Confocal microscopy confirmed the cell membrane localization of TGM2 and showed focal co-localization of TGM2 and integrin–α5 in control and OSM-treated SW756 cells grown on fibronectin (Figure 4B).


Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma.

Caffarel MM, Chattopadhyay A, Araujo AM, Bauer J, Scarpini CG, Coleman N - J. Pathol. (2013)

Interactions between TGM2 and integrin–α5β1 in cervical SCC cells. (A) Flow-cytometric quantification of cell-surface TGM2 expressed by SW756 and CaSki, comparing cells treated with OSM for 48 h (OSM) with control cells treated with vehicle only (C). Negative control cells were stained with an isotype control IgG. (B) Immunofluorescence for TGM2 (green) and integrin–α5 (red) in SW756 cells treated with vehicle (control) or OSM (48 h) for 48 h, showing focal co-localization (yellow); scale bars = 10 µm. (C) Immunoprecipitation showing physical interaction between TGM2 and integrin–α5β1 in SW756 and CaSki. Cells cultured on fibronectin-coated plates and treated with vehicle (C) or OSM (OSM) for 48 h were immunoprecipitated with anti-integrin–α5β1 antibodies or with IgG isotype-matched control antibodies. The immunoprecipitated (IP) and unbound (UNB) fractions, together with whole-cell lysates, were then analysed by western blotting, using antibodies against TGM2 (top row), integrin–α5 (ITGA5; middle row) and integrin–β1 (ITGB1; bottom row). The anti-integrin–α5β1 antibody precipitated TGM2 in both cell lines (cf lanes 3 and 4 versus 7 and 8 for each panel), with a stronger band for SW756 than for CaSki. Increased levels of precipitated protein were seen in OSM-treated cells (cf lanes 4 versus 3 in each panel). (D) Western blot showing levels of Y397-phosphorylated Fak (P-Fak) and total Fak in SW756 and CaSki cells cultured on fibronectin-coated plates and treated with OSM or vehicle (C) for 48 h
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4288975&req=5

fig04: Interactions between TGM2 and integrin–α5β1 in cervical SCC cells. (A) Flow-cytometric quantification of cell-surface TGM2 expressed by SW756 and CaSki, comparing cells treated with OSM for 48 h (OSM) with control cells treated with vehicle only (C). Negative control cells were stained with an isotype control IgG. (B) Immunofluorescence for TGM2 (green) and integrin–α5 (red) in SW756 cells treated with vehicle (control) or OSM (48 h) for 48 h, showing focal co-localization (yellow); scale bars = 10 µm. (C) Immunoprecipitation showing physical interaction between TGM2 and integrin–α5β1 in SW756 and CaSki. Cells cultured on fibronectin-coated plates and treated with vehicle (C) or OSM (OSM) for 48 h were immunoprecipitated with anti-integrin–α5β1 antibodies or with IgG isotype-matched control antibodies. The immunoprecipitated (IP) and unbound (UNB) fractions, together with whole-cell lysates, were then analysed by western blotting, using antibodies against TGM2 (top row), integrin–α5 (ITGA5; middle row) and integrin–β1 (ITGB1; bottom row). The anti-integrin–α5β1 antibody precipitated TGM2 in both cell lines (cf lanes 3 and 4 versus 7 and 8 for each panel), with a stronger band for SW756 than for CaSki. Increased levels of precipitated protein were seen in OSM-treated cells (cf lanes 4 versus 3 in each panel). (D) Western blot showing levels of Y397-phosphorylated Fak (P-Fak) and total Fak in SW756 and CaSki cells cultured on fibronectin-coated plates and treated with OSM or vehicle (C) for 48 h
Mentions: By flow cytometry, we observed that OSM treatment significantly increased expression of integrin–α5β1 dimers at the cell surface in both SW756 and CaSki (Figure 3B). The function of TGM2 relates to its subcellular location, with cell surface protein being responsible for interactions with integrins 8. Flow cytometry demonstrated the presence of TGM2 on the cell surface of both SW756 and CaSki (Figure 4A). OSM treatment increased the levels of membrane-associated TGM2, although the differences did not reach statistical significance. Confocal microscopy confirmed the cell membrane localization of TGM2 and showed focal co-localization of TGM2 and integrin–α5 in control and OSM-treated SW756 cells grown on fibronectin (Figure 4B).

Bottom Line: Cervical SCC cells that over-express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro-malignant effects, including increased cell migration and invasiveness.Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in SCC cells.We conclude that an OSMR/TGM2/integrin-α5β1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, UK.

Show MeSH
Related in: MedlinePlus