Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.
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.
Affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298.Show MeSH
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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.
Affiliation: Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298.