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TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion.

Osborne LD, Li GZ, How T, O'Brien ET, Blobe GC, Superfine R, Mythreye K - Mol. Biol. Cell (2014)

Bottom Line: Recent studies implicate a role for cell mechanics in cancer progression.Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response.Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

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LARG and GEF-H1 overexpression after EMT partially rescues cell stiffness and stiffness response and attenuates migration and invasion. (A) NMuMGs were treated with 100 pM TGF-β for 48 h. Cell were transfected with plasmid containing LARG-GFP or GEF-H1-GFP for the final 16–24 h of treatment. Quantifications are shown normalized to β-actin and relative to control, confirming protein expression. (B) Average cell stiffness (G1) for cells transfected with GFP vector control (n = 65), TGF-β and GFP vector control (n = 55), and GFP-DNA constructs to overexpress GEF-H1 (n = 32) or LARG (n = 54). *p < 0.05 denotes stiffness difference relative to cells treated with TGF-β and GFP vector control. Error bars represent SEM; data were collected from three independent experiments. (C) Average force response at pulls 2 (G2/G1) and 8 (G8/G1) for NMuMG cells transfected with GFP vector control (n = 25), TGF-β and GFP vector control (n = 15), and GFP-DNA constructs to overexpress GEF-H1 (n = 16) or LARG (n = 23). #p < 0.05 denotes stiffness difference from G1; *p < 0.05 denotes stiffness response difference relative to cells treated with TGF-β and GFP vector control. Error bars represent SEM; data were collected from three independent experiments. (D, E) Average migration and invasion for cells treated with TGF-β and transfected with plasmid containing empty vector control (GFP), LARG-GFP, or GEF-H1 GFP for the final 16–24 h of treatment and sorted by flow cytometry for GFP expression. Quantifications given as fold migration or invasion relative to control (n = 3). *p < 0.05.
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Figure 8: LARG and GEF-H1 overexpression after EMT partially rescues cell stiffness and stiffness response and attenuates migration and invasion. (A) NMuMGs were treated with 100 pM TGF-β for 48 h. Cell were transfected with plasmid containing LARG-GFP or GEF-H1-GFP for the final 16–24 h of treatment. Quantifications are shown normalized to β-actin and relative to control, confirming protein expression. (B) Average cell stiffness (G1) for cells transfected with GFP vector control (n = 65), TGF-β and GFP vector control (n = 55), and GFP-DNA constructs to overexpress GEF-H1 (n = 32) or LARG (n = 54). *p < 0.05 denotes stiffness difference relative to cells treated with TGF-β and GFP vector control. Error bars represent SEM; data were collected from three independent experiments. (C) Average force response at pulls 2 (G2/G1) and 8 (G8/G1) for NMuMG cells transfected with GFP vector control (n = 25), TGF-β and GFP vector control (n = 15), and GFP-DNA constructs to overexpress GEF-H1 (n = 16) or LARG (n = 23). #p < 0.05 denotes stiffness difference from G1; *p < 0.05 denotes stiffness response difference relative to cells treated with TGF-β and GFP vector control. Error bars represent SEM; data were collected from three independent experiments. (D, E) Average migration and invasion for cells treated with TGF-β and transfected with plasmid containing empty vector control (GFP), LARG-GFP, or GEF-H1 GFP for the final 16–24 h of treatment and sorted by flow cytometry for GFP expression. Quantifications given as fold migration or invasion relative to control (n = 3). *p < 0.05.

Mentions: Our results indicate that LARG and GEF-H1 are necessary mediators of stiffness (Figure 7B) and stiffness response (Figure 7C). To examine whether down-regulation of LARG and GEF-H1 was necessary and sufficient to restore post-EMT loss of stiffness and stiffness response phenotypes, we rescued GEF expression after EMT induction and performed mechanical and invasion assays. Post-EMT NMuMG cells were transfected with plasmids containing cDNA to encode GFP-tagged LARG and GEF-H1 to rescue the reduced expression (Figure 8A). Mechanical measurements were performed only on LARG- or GEF-H1–expressing cells, as identified by GFP expression. We found that restoring LARG or GEF-H1 expression rescued the 50% post-EMT reduction in cell stiffness to 70 and 80% of the GFP-control stiffness, respectively (Figure 8B). Similarly, restoring LARG or GEF-H1 expression rescues the 30% post-EMT reduction in stiffening response to 90% of the response observed in GFP-control cells (Figure 8C).


TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion.

Osborne LD, Li GZ, How T, O'Brien ET, Blobe GC, Superfine R, Mythreye K - Mol. Biol. Cell (2014)

LARG and GEF-H1 overexpression after EMT partially rescues cell stiffness and stiffness response and attenuates migration and invasion. (A) NMuMGs were treated with 100 pM TGF-β for 48 h. Cell were transfected with plasmid containing LARG-GFP or GEF-H1-GFP for the final 16–24 h of treatment. Quantifications are shown normalized to β-actin and relative to control, confirming protein expression. (B) Average cell stiffness (G1) for cells transfected with GFP vector control (n = 65), TGF-β and GFP vector control (n = 55), and GFP-DNA constructs to overexpress GEF-H1 (n = 32) or LARG (n = 54). *p < 0.05 denotes stiffness difference relative to cells treated with TGF-β and GFP vector control. Error bars represent SEM; data were collected from three independent experiments. (C) Average force response at pulls 2 (G2/G1) and 8 (G8/G1) for NMuMG cells transfected with GFP vector control (n = 25), TGF-β and GFP vector control (n = 15), and GFP-DNA constructs to overexpress GEF-H1 (n = 16) or LARG (n = 23). #p < 0.05 denotes stiffness difference from G1; *p < 0.05 denotes stiffness response difference relative to cells treated with TGF-β and GFP vector control. Error bars represent SEM; data were collected from three independent experiments. (D, E) Average migration and invasion for cells treated with TGF-β and transfected with plasmid containing empty vector control (GFP), LARG-GFP, or GEF-H1 GFP for the final 16–24 h of treatment and sorted by flow cytometry for GFP expression. Quantifications given as fold migration or invasion relative to control (n = 3). *p < 0.05.
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Figure 8: LARG and GEF-H1 overexpression after EMT partially rescues cell stiffness and stiffness response and attenuates migration and invasion. (A) NMuMGs were treated with 100 pM TGF-β for 48 h. Cell were transfected with plasmid containing LARG-GFP or GEF-H1-GFP for the final 16–24 h of treatment. Quantifications are shown normalized to β-actin and relative to control, confirming protein expression. (B) Average cell stiffness (G1) for cells transfected with GFP vector control (n = 65), TGF-β and GFP vector control (n = 55), and GFP-DNA constructs to overexpress GEF-H1 (n = 32) or LARG (n = 54). *p < 0.05 denotes stiffness difference relative to cells treated with TGF-β and GFP vector control. Error bars represent SEM; data were collected from three independent experiments. (C) Average force response at pulls 2 (G2/G1) and 8 (G8/G1) for NMuMG cells transfected with GFP vector control (n = 25), TGF-β and GFP vector control (n = 15), and GFP-DNA constructs to overexpress GEF-H1 (n = 16) or LARG (n = 23). #p < 0.05 denotes stiffness difference from G1; *p < 0.05 denotes stiffness response difference relative to cells treated with TGF-β and GFP vector control. Error bars represent SEM; data were collected from three independent experiments. (D, E) Average migration and invasion for cells treated with TGF-β and transfected with plasmid containing empty vector control (GFP), LARG-GFP, or GEF-H1 GFP for the final 16–24 h of treatment and sorted by flow cytometry for GFP expression. Quantifications given as fold migration or invasion relative to control (n = 3). *p < 0.05.
Mentions: Our results indicate that LARG and GEF-H1 are necessary mediators of stiffness (Figure 7B) and stiffness response (Figure 7C). To examine whether down-regulation of LARG and GEF-H1 was necessary and sufficient to restore post-EMT loss of stiffness and stiffness response phenotypes, we rescued GEF expression after EMT induction and performed mechanical and invasion assays. Post-EMT NMuMG cells were transfected with plasmids containing cDNA to encode GFP-tagged LARG and GEF-H1 to rescue the reduced expression (Figure 8A). Mechanical measurements were performed only on LARG- or GEF-H1–expressing cells, as identified by GFP expression. We found that restoring LARG or GEF-H1 expression rescued the 50% post-EMT reduction in cell stiffness to 70 and 80% of the GFP-control stiffness, respectively (Figure 8B). Similarly, restoring LARG or GEF-H1 expression rescues the 30% post-EMT reduction in stiffening response to 90% of the response observed in GFP-control cells (Figure 8C).

Bottom Line: Recent studies implicate a role for cell mechanics in cancer progression.Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response.Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

Show MeSH
Related in: MedlinePlus