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Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

Lechuga S, Baranwal S, Li C, Naydenov NG, Kuemmerle JF, Dugina V, Chaponnier C, Ivanov AI - Mol. Biol. Cell (2014)

Bottom Line: Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood.Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B.Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298.

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Upregulation of EMyT markers in γ-CYA-depleted cells depends on formin-mediated actin polymerization. (A) A549 cells were transfected with control or β-CYA– or γ-CYA–specific siRNA duplexes, and expression of different actin-polymerizing proteins was analyzed by immunoblotting. (B, C) Control and γ-CYA–depleted A549 cells were treated for 24 h with either vehicle or pharmacological inhibitors of Arp2/3 complex (CK-666) and formin-dependent actin polymerization (SMIFH2). Effects of the inhibitors on induction of EMyT marker were determined by immunoblotting. (D, E) A549 cells were subjected to sequential transfections with one of the following siRNA pairs: control–control, FHOD1–control, control–γ-CYA, and FHOD1–γ-CYA. Expression of targeted proteins and EMyT markers was determined by immunoblotting on day 3 after the second transfection. Data are presented as mean ± SE (n = 3); *p < 0.05, **p < 0.005, ***p < 0.0005.
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Figure 7: Upregulation of EMyT markers in γ-CYA-depleted cells depends on formin-mediated actin polymerization. (A) A549 cells were transfected with control or β-CYA– or γ-CYA–specific siRNA duplexes, and expression of different actin-polymerizing proteins was analyzed by immunoblotting. (B, C) Control and γ-CYA–depleted A549 cells were treated for 24 h with either vehicle or pharmacological inhibitors of Arp2/3 complex (CK-666) and formin-dependent actin polymerization (SMIFH2). Effects of the inhibitors on induction of EMyT marker were determined by immunoblotting. (D, E) A549 cells were subjected to sequential transfections with one of the following siRNA pairs: control–control, FHOD1–control, control–γ-CYA, and FHOD1–γ-CYA. Expression of targeted proteins and EMyT markers was determined by immunoblotting on day 3 after the second transfection. Data are presented as mean ± SE (n = 3); *p < 0.05, **p < 0.005, ***p < 0.0005.

Mentions: Because increased actin polymerization appears to be involved in the EMyT of γ-CYA–depleted epithelial cells, we investigated mechanisms underlying this process. A large number of actin-binding proteins can stimulate polymerization of either linear or branched actin filaments (Campellone and Welch, 2010). Immunoblotting of several well-characterized actin-polymerizing factors showed a selective increase in the expression of formin homology 2 domain containing 1 (FHOD1) in γ-CYA–depleted A549 cells (Figure 7A). According to densitometric analysis, FHOD1 protein level increased (3.0 ± 0.8)- and (2.5 ± 0.6)-fold in cells treated with γ-CYA siRNA duplex 1 and 2, respectively (n = 3, p < 0.05). To examine the involvement of different modes of actin polymerization in EMyT induction, we treated γ-CYA–depleted cells for 24 h with a pharmacological inhibitor of formins, SMIFH2 (50 μM; Rizvi et al., 2009), an inhibitor of Arp2/3 polymerization, CK-666 (50 μM; Nolen et al., 2009), or vehicle. The formin inhibitor significantly suppressed induction of all EMyT markers, whereas inhibition of the Arp2/3 complex was ineffective (Figure 7B). To investigate the specific role of FHOD1, we compared the effects of γ-CYA and FHOD1/γ-CYA knockdowns on EMyT induction. Depletion of FHOD1 only partially reversed the induction of α-SMA and CNN-1 without having significant effects on SM-22 and L-Cald expression (Figure 7C). These data indicate that up-regulation of FHOD1 alone does not explain the entire biochemical signature of EMyT of γ-CYA–depleted epithelial cells, which could be regulated by additional members of the formin family or other actin-binding proteins.


Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

Lechuga S, Baranwal S, Li C, Naydenov NG, Kuemmerle JF, Dugina V, Chaponnier C, Ivanov AI - Mol. Biol. Cell (2014)

Upregulation of EMyT markers in γ-CYA-depleted cells depends on formin-mediated actin polymerization. (A) A549 cells were transfected with control or β-CYA– or γ-CYA–specific siRNA duplexes, and expression of different actin-polymerizing proteins was analyzed by immunoblotting. (B, C) Control and γ-CYA–depleted A549 cells were treated for 24 h with either vehicle or pharmacological inhibitors of Arp2/3 complex (CK-666) and formin-dependent actin polymerization (SMIFH2). Effects of the inhibitors on induction of EMyT marker were determined by immunoblotting. (D, E) A549 cells were subjected to sequential transfections with one of the following siRNA pairs: control–control, FHOD1–control, control–γ-CYA, and FHOD1–γ-CYA. Expression of targeted proteins and EMyT markers was determined by immunoblotting on day 3 after the second transfection. Data are presented as mean ± SE (n = 3); *p < 0.05, **p < 0.005, ***p < 0.0005.
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Figure 7: Upregulation of EMyT markers in γ-CYA-depleted cells depends on formin-mediated actin polymerization. (A) A549 cells were transfected with control or β-CYA– or γ-CYA–specific siRNA duplexes, and expression of different actin-polymerizing proteins was analyzed by immunoblotting. (B, C) Control and γ-CYA–depleted A549 cells were treated for 24 h with either vehicle or pharmacological inhibitors of Arp2/3 complex (CK-666) and formin-dependent actin polymerization (SMIFH2). Effects of the inhibitors on induction of EMyT marker were determined by immunoblotting. (D, E) A549 cells were subjected to sequential transfections with one of the following siRNA pairs: control–control, FHOD1–control, control–γ-CYA, and FHOD1–γ-CYA. Expression of targeted proteins and EMyT markers was determined by immunoblotting on day 3 after the second transfection. Data are presented as mean ± SE (n = 3); *p < 0.05, **p < 0.005, ***p < 0.0005.
Mentions: Because increased actin polymerization appears to be involved in the EMyT of γ-CYA–depleted epithelial cells, we investigated mechanisms underlying this process. A large number of actin-binding proteins can stimulate polymerization of either linear or branched actin filaments (Campellone and Welch, 2010). Immunoblotting of several well-characterized actin-polymerizing factors showed a selective increase in the expression of formin homology 2 domain containing 1 (FHOD1) in γ-CYA–depleted A549 cells (Figure 7A). According to densitometric analysis, FHOD1 protein level increased (3.0 ± 0.8)- and (2.5 ± 0.6)-fold in cells treated with γ-CYA siRNA duplex 1 and 2, respectively (n = 3, p < 0.05). To examine the involvement of different modes of actin polymerization in EMyT induction, we treated γ-CYA–depleted cells for 24 h with a pharmacological inhibitor of formins, SMIFH2 (50 μM; Rizvi et al., 2009), an inhibitor of Arp2/3 polymerization, CK-666 (50 μM; Nolen et al., 2009), or vehicle. The formin inhibitor significantly suppressed induction of all EMyT markers, whereas inhibition of the Arp2/3 complex was ineffective (Figure 7B). To investigate the specific role of FHOD1, we compared the effects of γ-CYA and FHOD1/γ-CYA knockdowns on EMyT induction. Depletion of FHOD1 only partially reversed the induction of α-SMA and CNN-1 without having significant effects on SM-22 and L-Cald expression (Figure 7C). These data indicate that up-regulation of FHOD1 alone does not explain the entire biochemical signature of EMyT of γ-CYA–depleted epithelial cells, which could be regulated by additional members of the formin family or other actin-binding proteins.

Bottom Line: Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood.Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B.Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction.

View Article: PubMed Central - PubMed

Affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298.

Show MeSH
Related in: MedlinePlus