Limits...
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.

Show MeSH

Related in: MedlinePlus

LARG and GEF-H1 knockdowns decrease cell stiffness and stiffness response to force and increase cell migration and invasion. (A) NMuMG cells were transfected with siRNA against LARG, GEF-H1, both, or p114 for 48 h. Quantifications are given normalized to β-actin and relative to control, confirming knockdown of protein expression. (B) Average cell stiffness (G1) for NMuMG cells treated with siRNA control (n = 88), siRNA targeting p114 (n = 70), GEF-H1 (n = 90), LARG (n = 85), GEF-H1 + LARG (n = 100), and NMuMG cells treated with TGF-β and siRNA control (n = 69). *p < 0.01. Error bars represent SEM; data were collected from three independent experiments. (C) Average stiffness response at pulls 2 (G2/G1) and 8 (G8/G1) for NMuMG cells treated with siRNA control (n = 20), siRNA targeting p114 (n = 41), GEF-H1 (n = 19), LARG (n = 21), GEF-H1 + LARG (n = 30), and NMuMG cells treated with TGF-β and siRNA control (n = 19). #p < 0.05 stiffness difference from G1; *p < 0.05 stiffness response difference relative to cells treated with siRNA control. Error bars represent SEM; data were collected from three independent experiments. (D, E) Average migration and invasion for NMuMG cells treated with siRNA as indicated: control, GEF-H1, LARG, both, or p114 for 36 h before plating onto uncoated or Matrigel-coated Transwell filters. *p < 0.05. Error bars represent SEM; data represent mean of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230614&req=5

Figure 7: LARG and GEF-H1 knockdowns decrease cell stiffness and stiffness response to force and increase cell migration and invasion. (A) NMuMG cells were transfected with siRNA against LARG, GEF-H1, both, or p114 for 48 h. Quantifications are given normalized to β-actin and relative to control, confirming knockdown of protein expression. (B) Average cell stiffness (G1) for NMuMG cells treated with siRNA control (n = 88), siRNA targeting p114 (n = 70), GEF-H1 (n = 90), LARG (n = 85), GEF-H1 + LARG (n = 100), and NMuMG cells treated with TGF-β and siRNA control (n = 69). *p < 0.01. Error bars represent SEM; data were collected from three independent experiments. (C) Average stiffness response at pulls 2 (G2/G1) and 8 (G8/G1) for NMuMG cells treated with siRNA control (n = 20), siRNA targeting p114 (n = 41), GEF-H1 (n = 19), LARG (n = 21), GEF-H1 + LARG (n = 30), and NMuMG cells treated with TGF-β and siRNA control (n = 19). #p < 0.05 stiffness difference from G1; *p < 0.05 stiffness response difference relative to cells treated with siRNA control. Error bars represent SEM; data were collected from three independent experiments. (D, E) Average migration and invasion for NMuMG cells treated with siRNA as indicated: control, GEF-H1, LARG, both, or p114 for 36 h before plating onto uncoated or Matrigel-coated Transwell filters. *p < 0.05. Error bars represent SEM; data represent mean of three independent experiments.

Mentions: EMT marks the physical initiation of cancer progression as a cell detaches from the primary tumor and invades the surrounding stromal space. Alterations in the RhoA pathway have been implicated in a variety of cancers (Simpson et al., 2004; Vega et al., 2011). However, whereas RhoA activation in some cancers is associated with increased invasion (Liao et al., 2012), in others, RhoA activation inhibits cell invasion (Bellovin et al., 2006). In addition, RhoA and its associated GEFs LARG and GEF-H1 have been shown to regulate the stiffening response to force applied on integrins (Guilluy et al., 2011). On the basis of these and our findings that LARG and GEF-H1 expression and force-dependent recruitment are reduced in multiple EMT models (Figures 4A and 5A) and that ALK5 inhibition reverses the TGF-β–mediated reduction in cell mechanics, we examined whether LARG and GEF-H1 down-regulation were sufficient for decreased stiffness and stiffening response to force. Using small interfering RNA (siRNA) to reduce GEF expression in NMuMG epithelial-state cells (Figure 7A), we found that silencing LARG, GEF-H1, or both significantly decreased cellular stiffness (Figure 7B) and stiffening response compared with control siRNA–treated cells (Figure 7C). In contrast, siRNA to p114 had no significant effect on stiffness (Figure 7B) or suppression of the stiffening response to force (Figure 7C), suggesting a specific role for LARG and GEF-H1 in determining these mechanical properties.


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 knockdowns decrease cell stiffness and stiffness response to force and increase cell migration and invasion. (A) NMuMG cells were transfected with siRNA against LARG, GEF-H1, both, or p114 for 48 h. Quantifications are given normalized to β-actin and relative to control, confirming knockdown of protein expression. (B) Average cell stiffness (G1) for NMuMG cells treated with siRNA control (n = 88), siRNA targeting p114 (n = 70), GEF-H1 (n = 90), LARG (n = 85), GEF-H1 + LARG (n = 100), and NMuMG cells treated with TGF-β and siRNA control (n = 69). *p < 0.01. Error bars represent SEM; data were collected from three independent experiments. (C) Average stiffness response at pulls 2 (G2/G1) and 8 (G8/G1) for NMuMG cells treated with siRNA control (n = 20), siRNA targeting p114 (n = 41), GEF-H1 (n = 19), LARG (n = 21), GEF-H1 + LARG (n = 30), and NMuMG cells treated with TGF-β and siRNA control (n = 19). #p < 0.05 stiffness difference from G1; *p < 0.05 stiffness response difference relative to cells treated with siRNA control. Error bars represent SEM; data were collected from three independent experiments. (D, E) Average migration and invasion for NMuMG cells treated with siRNA as indicated: control, GEF-H1, LARG, both, or p114 for 36 h before plating onto uncoated or Matrigel-coated Transwell filters. *p < 0.05. Error bars represent SEM; data represent mean of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230614&req=5

Figure 7: LARG and GEF-H1 knockdowns decrease cell stiffness and stiffness response to force and increase cell migration and invasion. (A) NMuMG cells were transfected with siRNA against LARG, GEF-H1, both, or p114 for 48 h. Quantifications are given normalized to β-actin and relative to control, confirming knockdown of protein expression. (B) Average cell stiffness (G1) for NMuMG cells treated with siRNA control (n = 88), siRNA targeting p114 (n = 70), GEF-H1 (n = 90), LARG (n = 85), GEF-H1 + LARG (n = 100), and NMuMG cells treated with TGF-β and siRNA control (n = 69). *p < 0.01. Error bars represent SEM; data were collected from three independent experiments. (C) Average stiffness response at pulls 2 (G2/G1) and 8 (G8/G1) for NMuMG cells treated with siRNA control (n = 20), siRNA targeting p114 (n = 41), GEF-H1 (n = 19), LARG (n = 21), GEF-H1 + LARG (n = 30), and NMuMG cells treated with TGF-β and siRNA control (n = 19). #p < 0.05 stiffness difference from G1; *p < 0.05 stiffness response difference relative to cells treated with siRNA control. Error bars represent SEM; data were collected from three independent experiments. (D, E) Average migration and invasion for NMuMG cells treated with siRNA as indicated: control, GEF-H1, LARG, both, or p114 for 36 h before plating onto uncoated or Matrigel-coated Transwell filters. *p < 0.05. Error bars represent SEM; data represent mean of three independent experiments.
Mentions: EMT marks the physical initiation of cancer progression as a cell detaches from the primary tumor and invades the surrounding stromal space. Alterations in the RhoA pathway have been implicated in a variety of cancers (Simpson et al., 2004; Vega et al., 2011). However, whereas RhoA activation in some cancers is associated with increased invasion (Liao et al., 2012), in others, RhoA activation inhibits cell invasion (Bellovin et al., 2006). In addition, RhoA and its associated GEFs LARG and GEF-H1 have been shown to regulate the stiffening response to force applied on integrins (Guilluy et al., 2011). On the basis of these and our findings that LARG and GEF-H1 expression and force-dependent recruitment are reduced in multiple EMT models (Figures 4A and 5A) and that ALK5 inhibition reverses the TGF-β–mediated reduction in cell mechanics, we examined whether LARG and GEF-H1 down-regulation were sufficient for decreased stiffness and stiffening response to force. Using small interfering RNA (siRNA) to reduce GEF expression in NMuMG epithelial-state cells (Figure 7A), we found that silencing LARG, GEF-H1, or both significantly decreased cellular stiffness (Figure 7B) and stiffening response compared with control siRNA–treated cells (Figure 7C). In contrast, siRNA to p114 had no significant effect on stiffness (Figure 7B) or suppression of the stiffening response to force (Figure 7C), suggesting a specific role for LARG and GEF-H1 in determining these mechanical properties.

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