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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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Disassembly of intercellular junctions does not contribute to induction of EMyT in γ-CYA–depleted epithelial cells. A549 cells were transfected with either control or cytoplasmic actin isoform–specific siRNAs. The integrity of cell–cell contacts (A) and expression of junctional proteins (B) were examined on day 4 posttransfection. Arrows highlight disassembly of intercellular junctions in γ-CYA–depleted cells. (C) A549 cells were subjected to sequential transfections with one of the following siRNA pairs: control–control, E-cadherin–control, control–γ-CYA, and E-cadherin–γ-CYA. Expression of targeted proteins and EMyT markers was determined by immunoblotting on day 3 after the second transfection. (D) Control and γ-CYA–depleted A549 cells were either incubated in normal cell culture medium (+Ca2+) or subjected to 24 h of extracellular calcium depletion (–Ca2+) and analyzed for expression of EMyT markers.
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Figure 9: Disassembly of intercellular junctions does not contribute to induction of EMyT in γ-CYA–depleted epithelial cells. A549 cells were transfected with either control or cytoplasmic actin isoform–specific siRNAs. The integrity of cell–cell contacts (A) and expression of junctional proteins (B) were examined on day 4 posttransfection. Arrows highlight disassembly of intercellular junctions in γ-CYA–depleted cells. (C) A549 cells were subjected to sequential transfections with one of the following siRNA pairs: control–control, E-cadherin–control, control–γ-CYA, and E-cadherin–γ-CYA. Expression of targeted proteins and EMyT markers was determined by immunoblotting on day 3 after the second transfection. (D) Control and γ-CYA–depleted A549 cells were either incubated in normal cell culture medium (+Ca2+) or subjected to 24 h of extracellular calcium depletion (–Ca2+) and analyzed for expression of EMyT markers.

Mentions: Next we asked whether up-regulation of contractile proteins can be causally linked to the loss of some epithelial features in γ-CYA–depleted A549 cells. Considering the compelling data that disruption of cell–cell junctions is capable of activating Rho and stimulating the EMyT of renal and mammary epithelia (Fan et al., 2007; Speight et al., 2013), we investigated the effects of junctional disassembly on transdifferentiation of γ-CYA depleted cells. First, we examined the integrity of cell–cell contacts along with the expression of major junctional proteins. Immunofluorescence analysis showed dramatic disruption of E-cadherin–based adherens junctions (AJs) and zonula occludens 1 (ZO-1)–based tight junctions (TJs) in γ-CYA–deficient A549 cells (Figure 9A, arrows). Furthermore, γ-CYA knockdown markedly decreased protein expression of E-cadherin and β-catenin and upregulated ZO-1 levels in A549 cells (Figure 9B). These data suggest that in poorly differentiated epithelial cells, such as A549, γ-CYA controls the integrity of both AJs and TJs, which contrasts with its functions in well-differentiated colonic epithelium, where γ-CYA was shown to selectively regulate TJ assembly (Baranwal et al., 2012). If down-regulation of cell–cell contacts contributes to transcriptional reprogramming of A549 cells, forceful junctional disassembly should accelerate expression of EMyT markers either in control or γ-CYA–depleted epithelial cells. This hypothesis was tested using two different approaches; one involved siRNA-mediated down-regulation of E-cadherin expression, and the other involved global disassembly of epithelial junctions by extracellular calcium depletion (Ivanov et al., 2004). Neither approach resulted in induction of EMyT in control A549 cells (Figure 9, C and D). Furthermore, in contrast to our prediction, loss of E-cadherin inhibited rather than accelerated expression of some contractile proteins (α-SMA and SM-22) in γ-CYA–depleted cells, whereas calcium depletion was entirely ineffective (Figure 9, C and D). Together these data strongly suggest that disruption of epithelial junctions plays no role in transcriptional reprogramming of γ-CYA–depleted cells.


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)

Disassembly of intercellular junctions does not contribute to induction of EMyT in γ-CYA–depleted epithelial cells. A549 cells were transfected with either control or cytoplasmic actin isoform–specific siRNAs. The integrity of cell–cell contacts (A) and expression of junctional proteins (B) were examined on day 4 posttransfection. Arrows highlight disassembly of intercellular junctions in γ-CYA–depleted cells. (C) A549 cells were subjected to sequential transfections with one of the following siRNA pairs: control–control, E-cadherin–control, control–γ-CYA, and E-cadherin–γ-CYA. Expression of targeted proteins and EMyT markers was determined by immunoblotting on day 3 after the second transfection. (D) Control and γ-CYA–depleted A549 cells were either incubated in normal cell culture medium (+Ca2+) or subjected to 24 h of extracellular calcium depletion (–Ca2+) and analyzed for expression of EMyT markers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 9: Disassembly of intercellular junctions does not contribute to induction of EMyT in γ-CYA–depleted epithelial cells. A549 cells were transfected with either control or cytoplasmic actin isoform–specific siRNAs. The integrity of cell–cell contacts (A) and expression of junctional proteins (B) were examined on day 4 posttransfection. Arrows highlight disassembly of intercellular junctions in γ-CYA–depleted cells. (C) A549 cells were subjected to sequential transfections with one of the following siRNA pairs: control–control, E-cadherin–control, control–γ-CYA, and E-cadherin–γ-CYA. Expression of targeted proteins and EMyT markers was determined by immunoblotting on day 3 after the second transfection. (D) Control and γ-CYA–depleted A549 cells were either incubated in normal cell culture medium (+Ca2+) or subjected to 24 h of extracellular calcium depletion (–Ca2+) and analyzed for expression of EMyT markers.
Mentions: Next we asked whether up-regulation of contractile proteins can be causally linked to the loss of some epithelial features in γ-CYA–depleted A549 cells. Considering the compelling data that disruption of cell–cell junctions is capable of activating Rho and stimulating the EMyT of renal and mammary epithelia (Fan et al., 2007; Speight et al., 2013), we investigated the effects of junctional disassembly on transdifferentiation of γ-CYA depleted cells. First, we examined the integrity of cell–cell contacts along with the expression of major junctional proteins. Immunofluorescence analysis showed dramatic disruption of E-cadherin–based adherens junctions (AJs) and zonula occludens 1 (ZO-1)–based tight junctions (TJs) in γ-CYA–deficient A549 cells (Figure 9A, arrows). Furthermore, γ-CYA knockdown markedly decreased protein expression of E-cadherin and β-catenin and upregulated ZO-1 levels in A549 cells (Figure 9B). These data suggest that in poorly differentiated epithelial cells, such as A549, γ-CYA controls the integrity of both AJs and TJs, which contrasts with its functions in well-differentiated colonic epithelium, where γ-CYA was shown to selectively regulate TJ assembly (Baranwal et al., 2012). If down-regulation of cell–cell contacts contributes to transcriptional reprogramming of A549 cells, forceful junctional disassembly should accelerate expression of EMyT markers either in control or γ-CYA–depleted epithelial cells. This hypothesis was tested using two different approaches; one involved siRNA-mediated down-regulation of E-cadherin expression, and the other involved global disassembly of epithelial junctions by extracellular calcium depletion (Ivanov et al., 2004). Neither approach resulted in induction of EMyT in control A549 cells (Figure 9, C and D). Furthermore, in contrast to our prediction, loss of E-cadherin inhibited rather than accelerated expression of some contractile proteins (α-SMA and SM-22) in γ-CYA–depleted cells, whereas calcium depletion was entirely ineffective (Figure 9, C and D). Together these data strongly suggest that disruption of epithelial junctions plays no role in transcriptional reprogramming of γ-CYA–depleted 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.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus