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

TGF-β promotes proteasome degradation of LARG and GEF-H1 during EMT via Alk5. (A) Indicated cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor), 10 μM U0126 (MEK inhibitor), or dimethyl sulfoxide (DMSO; negative control), followed by treatment with 100 pM TGF-β for 48 h. (B) NMuMG cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor) or DMSO, followed by treatment with 100 pM TGF-β for 48 h or DMSO. Average cell stiffness for DMSO (n = 55), DMSO and TGF-β (n = 84), and SB-431542 and TGF-β (n = 81) populations. *p < 0.01. Error bars represent SEM; data were collected from three independent experiments. (C) NMuMG cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor) or DMSO, followed by treatment with 100 pM TGF-β for 48 h or DMSO. Average stiffness response for DMSO (n = 31), DMSO and TGF-β (n = 20), and SB-431542 and TGF-β (n = 26) populations. *p < 0.05. Error bars represent SEM; data were collected from three independent experiments. (D) NMuMG cells transfected with GFP-tagged LARG or GEF-H1cDNA lacking the 3′-UTR were treated with TGF-β for 48 h. Lysates were immunoblotted to determine expression of endogenous and exogenous GFP-tagged LARG/GEFH1. (E) TGF-β treatment does not reduce mRNA levels of LARG or GEF-H1. mRNA levels determined by quantitative PCR were normalized to glyceraldehyde-3-phosphate dehydrogenase. Error bars represent SEM. The dashed line denotes untreated. (F) TGF-β promotes proteasome degradation of LARG and GEF-H1. (D) OVCA420 cells were treated for 72 h with TGF-β and with 10 and 20 μM MG-132 or DMSO as control. (E) NMuMG cells treated with TGF-β for 48 h and 10 μM MG-132 or DMSO as control for the last 16 h of treatment for both D and E. Representative of at least three experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230614&req=5

Figure 6: TGF-β promotes proteasome degradation of LARG and GEF-H1 during EMT via Alk5. (A) Indicated cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor), 10 μM U0126 (MEK inhibitor), or dimethyl sulfoxide (DMSO; negative control), followed by treatment with 100 pM TGF-β for 48 h. (B) NMuMG cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor) or DMSO, followed by treatment with 100 pM TGF-β for 48 h or DMSO. Average cell stiffness for DMSO (n = 55), DMSO and TGF-β (n = 84), and SB-431542 and TGF-β (n = 81) populations. *p < 0.01. Error bars represent SEM; data were collected from three independent experiments. (C) NMuMG cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor) or DMSO, followed by treatment with 100 pM TGF-β for 48 h or DMSO. Average stiffness response for DMSO (n = 31), DMSO and TGF-β (n = 20), and SB-431542 and TGF-β (n = 26) populations. *p < 0.05. Error bars represent SEM; data were collected from three independent experiments. (D) NMuMG cells transfected with GFP-tagged LARG or GEF-H1cDNA lacking the 3′-UTR were treated with TGF-β for 48 h. Lysates were immunoblotted to determine expression of endogenous and exogenous GFP-tagged LARG/GEFH1. (E) TGF-β treatment does not reduce mRNA levels of LARG or GEF-H1. mRNA levels determined by quantitative PCR were normalized to glyceraldehyde-3-phosphate dehydrogenase. Error bars represent SEM. The dashed line denotes untreated. (F) TGF-β promotes proteasome degradation of LARG and GEF-H1. (D) OVCA420 cells were treated for 72 h with TGF-β and with 10 and 20 μM MG-132 or DMSO as control. (E) NMuMG cells treated with TGF-β for 48 h and 10 μM MG-132 or DMSO as control for the last 16 h of treatment for both D and E. Representative of at least three experiments.

Mentions: TGF-β can regulate activation of the RhoA pathway via canonical (activin receptor–like kinase 5 [ALK5] dependent) and noncanonical (mitogen-activated protein kinase dependent) TGF-β signaling mechanisms (Bhowmick et al., 2001). To investigate how TGF-β regulates LARG and GEF-H1 expression during EMT, we used the ALK5 inhibitor SB-431542 (Alk et al., 2002) and found that ALK5 inhibition partially rescued TGF-β–mediated LARG and GEF-H1 protein down-regulation in both NMuMG and OVCA420 cells, indicating a requirement for ALK5 in maximal regulation of RhoGEF expression (Figure 6A). In contrast, blocking the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase pathway with U0126 (Alk et al., 2002), which can also mediate TGF-β responses (Xu et al., 2009), did not ameliorate TGF-β–dependent decreases in LARG and GEF-H1 levels in NMuMG cells (Figure 6A).


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)

TGF-β promotes proteasome degradation of LARG and GEF-H1 during EMT via Alk5. (A) Indicated cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor), 10 μM U0126 (MEK inhibitor), or dimethyl sulfoxide (DMSO; negative control), followed by treatment with 100 pM TGF-β for 48 h. (B) NMuMG cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor) or DMSO, followed by treatment with 100 pM TGF-β for 48 h or DMSO. Average cell stiffness for DMSO (n = 55), DMSO and TGF-β (n = 84), and SB-431542 and TGF-β (n = 81) populations. *p < 0.01. Error bars represent SEM; data were collected from three independent experiments. (C) NMuMG cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor) or DMSO, followed by treatment with 100 pM TGF-β for 48 h or DMSO. Average stiffness response for DMSO (n = 31), DMSO and TGF-β (n = 20), and SB-431542 and TGF-β (n = 26) populations. *p < 0.05. Error bars represent SEM; data were collected from three independent experiments. (D) NMuMG cells transfected with GFP-tagged LARG or GEF-H1cDNA lacking the 3′-UTR were treated with TGF-β for 48 h. Lysates were immunoblotted to determine expression of endogenous and exogenous GFP-tagged LARG/GEFH1. (E) TGF-β treatment does not reduce mRNA levels of LARG or GEF-H1. mRNA levels determined by quantitative PCR were normalized to glyceraldehyde-3-phosphate dehydrogenase. Error bars represent SEM. The dashed line denotes untreated. (F) TGF-β promotes proteasome degradation of LARG and GEF-H1. (D) OVCA420 cells were treated for 72 h with TGF-β and with 10 and 20 μM MG-132 or DMSO as control. (E) NMuMG cells treated with TGF-β for 48 h and 10 μM MG-132 or DMSO as control for the last 16 h of treatment for both D and E. Representative of at least three experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: TGF-β promotes proteasome degradation of LARG and GEF-H1 during EMT via Alk5. (A) Indicated cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor), 10 μM U0126 (MEK inhibitor), or dimethyl sulfoxide (DMSO; negative control), followed by treatment with 100 pM TGF-β for 48 h. (B) NMuMG cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor) or DMSO, followed by treatment with 100 pM TGF-β for 48 h or DMSO. Average cell stiffness for DMSO (n = 55), DMSO and TGF-β (n = 84), and SB-431542 and TGF-β (n = 81) populations. *p < 0.01. Error bars represent SEM; data were collected from three independent experiments. (C) NMuMG cells were pretreated for 1 h with 10 μM SB-431542 (ALK5 inhibitor) or DMSO, followed by treatment with 100 pM TGF-β for 48 h or DMSO. Average stiffness response for DMSO (n = 31), DMSO and TGF-β (n = 20), and SB-431542 and TGF-β (n = 26) populations. *p < 0.05. Error bars represent SEM; data were collected from three independent experiments. (D) NMuMG cells transfected with GFP-tagged LARG or GEF-H1cDNA lacking the 3′-UTR were treated with TGF-β for 48 h. Lysates were immunoblotted to determine expression of endogenous and exogenous GFP-tagged LARG/GEFH1. (E) TGF-β treatment does not reduce mRNA levels of LARG or GEF-H1. mRNA levels determined by quantitative PCR were normalized to glyceraldehyde-3-phosphate dehydrogenase. Error bars represent SEM. The dashed line denotes untreated. (F) TGF-β promotes proteasome degradation of LARG and GEF-H1. (D) OVCA420 cells were treated for 72 h with TGF-β and with 10 and 20 μM MG-132 or DMSO as control. (E) NMuMG cells treated with TGF-β for 48 h and 10 μM MG-132 or DMSO as control for the last 16 h of treatment for both D and E. Representative of at least three experiments.
Mentions: TGF-β can regulate activation of the RhoA pathway via canonical (activin receptor–like kinase 5 [ALK5] dependent) and noncanonical (mitogen-activated protein kinase dependent) TGF-β signaling mechanisms (Bhowmick et al., 2001). To investigate how TGF-β regulates LARG and GEF-H1 expression during EMT, we used the ALK5 inhibitor SB-431542 (Alk et al., 2002) and found that ALK5 inhibition partially rescued TGF-β–mediated LARG and GEF-H1 protein down-regulation in both NMuMG and OVCA420 cells, indicating a requirement for ALK5 in maximal regulation of RhoGEF expression (Figure 6A). In contrast, blocking the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase pathway with U0126 (Alk et al., 2002), which can also mediate TGF-β responses (Xu et al., 2009), did not ameliorate TGF-β–dependent decreases in LARG and GEF-H1 levels in NMuMG cells (Figure 6A).

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