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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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Downregulation of γ-CYA selectively stimulates expression of EMyT markers in lung epithelial cells. A549 epithelial cells were transfected with control or β-CYA– or γ-CYA–specific siRNA duplexes (D1 and D2), and expression of EMyT markers was analyzed by immunoblotting (A, B) and quantitative real-time RT-PCR (C) at different times after transfection. Immunoblots are quantified by densitometric analysis, and protein expression is calculated relative to the control siRNA–treated group. mRNA expression of all EMyT markers is normalized by the expression of a housekeeping gene. Data are presented as mean ± SE (n = 3); *p < 0.05, **p < 0.005, ***p < 0.0005.
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Figure 1: Downregulation of γ-CYA selectively stimulates expression of EMyT markers in lung epithelial cells. A549 epithelial cells were transfected with control or β-CYA– or γ-CYA–specific siRNA duplexes (D1 and D2), and expression of EMyT markers was analyzed by immunoblotting (A, B) and quantitative real-time RT-PCR (C) at different times after transfection. Immunoblots are quantified by densitometric analysis, and protein expression is calculated relative to the control siRNA–treated group. mRNA expression of all EMyT markers is normalized by the expression of a housekeeping gene. Data are presented as mean ± SE (n = 3); *p < 0.05, **p < 0.005, ***p < 0.0005.

Mentions: Because the actin cytoskeleton can regulate development of EMT by switching expression from epithelial-specific to mesenchyme-specific sets of cytoskeletal proteins (Zheng et al., 2008; Beach et al., 2011), we initially asked whether cytoplasmic actin isoforms can affect the development of EMT. To answer this question, we used A549 human lung epithelial cells treated with transforming growth factor-β (TGF-β), which represents a common in vitro model of EMT (Kasai et al., 2005). As expected, 48-h exposure to TGF-β (10 ng/ml) induced a prominent morphological change and biochemical signature characteristic of EMT, including elongated cell shape (Supplemental Figure S1A), the cadherin switch (down-regulation of E-cadherin and up-regulation of N-cadherin), and induction of mesenchymal protein markers such as vimentin and fibronectin (Supplemental Figure S1C). Knockdown of either β-CYA or γ-CYA using actin isoform–specific small interfering RNA (siRNA) SmartPools did not show consistent effects on TGF-β–dependent changes in the expression of epithelial and mesenchymal markers (Supplemental Figure S1C). Unexpectedly, loss of γ-CYA but not β-CYA induced marked expression of α-smooth muscle actin (α-SMA) in the absence of TGF-β (Supplemental Figure S1C). Because α-SMA induction was not accompanied by significant up-regulation of other mesenchymal markers such as N-cadherin and fibronectin, we hypothesized that γ-CYA depletion triggers a specific subset of EMT known as epithelial-to-myofibroblast transformation (EMyT). EMyT is characterized by induction of contractile cytoskeletal proteins abundant in myofibroblasts and smooth muscle cells (Sandbo and Dulin, 2011). To test this idea, we depleted either γ-CYA or β-CYA in A549 epithelial cells by using individual actin isoform–specific siRNA duplexes and subsequently examined expression of different EMyT markers on day 4 posttransfection. Transfection with different siRNA duplexes resulted in efficient and specific depletion of targeted actin isoforms, with >97 and 83% decrease in β-CYA and γ-CYA expression, respectively (Figure 1A and unpublished data). Of interest, neither knockdown significantly affected the total level of actin, due to compensatory up-regulation of the remaining CYA isoform (Figure 1A). Using densitometric analysis of control and β-CYA–depleted cell lysates (no α-SMA induction) probed with either actin isoform–specific or total-actin antibodies, we estimated that control A549 cells express ∼45% γ-CYA and 55% β-CYA.


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)

Downregulation of γ-CYA selectively stimulates expression of EMyT markers in lung epithelial cells. A549 epithelial cells were transfected with control or β-CYA– or γ-CYA–specific siRNA duplexes (D1 and D2), and expression of EMyT markers was analyzed by immunoblotting (A, B) and quantitative real-time RT-PCR (C) at different times after transfection. Immunoblots are quantified by densitometric analysis, and protein expression is calculated relative to the control siRNA–treated group. mRNA expression of all EMyT markers is normalized by the expression of a housekeeping gene. Data are presented as mean ± SE (n = 3); *p < 0.05, **p < 0.005, ***p < 0.0005.
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Figure 1: Downregulation of γ-CYA selectively stimulates expression of EMyT markers in lung epithelial cells. A549 epithelial cells were transfected with control or β-CYA– or γ-CYA–specific siRNA duplexes (D1 and D2), and expression of EMyT markers was analyzed by immunoblotting (A, B) and quantitative real-time RT-PCR (C) at different times after transfection. Immunoblots are quantified by densitometric analysis, and protein expression is calculated relative to the control siRNA–treated group. mRNA expression of all EMyT markers is normalized by the expression of a housekeeping gene. Data are presented as mean ± SE (n = 3); *p < 0.05, **p < 0.005, ***p < 0.0005.
Mentions: Because the actin cytoskeleton can regulate development of EMT by switching expression from epithelial-specific to mesenchyme-specific sets of cytoskeletal proteins (Zheng et al., 2008; Beach et al., 2011), we initially asked whether cytoplasmic actin isoforms can affect the development of EMT. To answer this question, we used A549 human lung epithelial cells treated with transforming growth factor-β (TGF-β), which represents a common in vitro model of EMT (Kasai et al., 2005). As expected, 48-h exposure to TGF-β (10 ng/ml) induced a prominent morphological change and biochemical signature characteristic of EMT, including elongated cell shape (Supplemental Figure S1A), the cadherin switch (down-regulation of E-cadherin and up-regulation of N-cadherin), and induction of mesenchymal protein markers such as vimentin and fibronectin (Supplemental Figure S1C). Knockdown of either β-CYA or γ-CYA using actin isoform–specific small interfering RNA (siRNA) SmartPools did not show consistent effects on TGF-β–dependent changes in the expression of epithelial and mesenchymal markers (Supplemental Figure S1C). Unexpectedly, loss of γ-CYA but not β-CYA induced marked expression of α-smooth muscle actin (α-SMA) in the absence of TGF-β (Supplemental Figure S1C). Because α-SMA induction was not accompanied by significant up-regulation of other mesenchymal markers such as N-cadherin and fibronectin, we hypothesized that γ-CYA depletion triggers a specific subset of EMT known as epithelial-to-myofibroblast transformation (EMyT). EMyT is characterized by induction of contractile cytoskeletal proteins abundant in myofibroblasts and smooth muscle cells (Sandbo and Dulin, 2011). To test this idea, we depleted either γ-CYA or β-CYA in A549 epithelial cells by using individual actin isoform–specific siRNA duplexes and subsequently examined expression of different EMyT markers on day 4 posttransfection. Transfection with different siRNA duplexes resulted in efficient and specific depletion of targeted actin isoforms, with >97 and 83% decrease in β-CYA and γ-CYA expression, respectively (Figure 1A and unpublished data). Of interest, neither knockdown significantly affected the total level of actin, due to compensatory up-regulation of the remaining CYA isoform (Figure 1A). Using densitometric analysis of control and β-CYA–depleted cell lysates (no α-SMA induction) probed with either actin isoform–specific or total-actin antibodies, we estimated that control A549 cells express ∼45% γ-CYA and 55% β-CYA.

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