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Downregulation of OPA3 is responsible for transforming growth factor-β-induced mitochondrial elongation and F-actin rearrangement in retinal pigment epithelial ARPE-19 cells.

Ryu SW, Yoon J, Yim N, Choi K, Choi C - PLoS ONE (2013)

Bottom Line: We also showed that Drp1 depletion increased cell length and induced rearrangement of F-actin.Depletion of Mfn1 blocked the increase in cell length during TGF-β-mediated EMT.These results collectively substantiate the involvement of mitochondrial dynamics in TGF-β-induced EMT.

View Article: PubMed Central - PubMed

Affiliation: Department of Bio and Brain Engineering, KAIST, Daejeon, Korea. ryus@kaist.ac.kr

ABSTRACT
Transforming growth factor-β signaling is known to be a key signaling pathway in the induction of epithelial-mesenchymal transition. However, the mechanism of TGF-β signaling in the modulation of EMT remains unclear. In this study, we found that TGF-β treatment resulted in elongation of mitochondria accompanied by induction of N-cadherin, vimentin, and F-actin in retinal pigment epithelial cells. Moreover, OPA3, which plays a crucial role in mitochondrial dynamics, was downregulated following TGF-β treatment. Suppression of TGF-β signaling using Smad2 siRNA prevented loss of OPA3 induced by TGF-β. Knockdown of OPA3 by siRNA and inducible shRNA significantly increased stress fiber levels, cell length, cell migration and mitochondrial elongation. In contrast, forced expression of OPA3 in ARPE-19 cells inhibited F-actin rearrangement and induced mitochondrial fragmentation. We also showed that Drp1 depletion increased cell length and induced rearrangement of F-actin. Depletion of Mfn1 blocked the increase in cell length during TGF-β-mediated EMT. These results collectively substantiate the involvement of mitochondrial dynamics in TGF-β-induced EMT.

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Functional changes in cells induced by knockdown of proteins involved in mitochondrial dynamics.(A and B) The mitochondrial fusion by Drp1 knockdown leads to an increase of cell length and F-actin. APRE-19 cells were transfected with Drp1 siRNA, Mfn1 siRNA, or control siRNA. Forty-eight hours after transfection, cells were treated withTGF-β for 48 h. Cells were fixed and stained with phalloidin-TRITC (A). Cell length was analyzed using confocal images (B). Data are the mean ± SD of three experiments, each with 100 cells per condition. (C) The mitochondrial fusion by Drp1 knockdown promotes cell migration induced by TGF-β. Cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were treated with TGF-β for the indicated periods of time. The migrated cells were counted. Data shown represent the average of three independent experiments. *P<0.05; ***P<0.0005.
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pone-0063495-g005: Functional changes in cells induced by knockdown of proteins involved in mitochondrial dynamics.(A and B) The mitochondrial fusion by Drp1 knockdown leads to an increase of cell length and F-actin. APRE-19 cells were transfected with Drp1 siRNA, Mfn1 siRNA, or control siRNA. Forty-eight hours after transfection, cells were treated withTGF-β for 48 h. Cells were fixed and stained with phalloidin-TRITC (A). Cell length was analyzed using confocal images (B). Data are the mean ± SD of three experiments, each with 100 cells per condition. (C) The mitochondrial fusion by Drp1 knockdown promotes cell migration induced by TGF-β. Cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were treated with TGF-β for the indicated periods of time. The migrated cells were counted. Data shown represent the average of three independent experiments. *P<0.05; ***P<0.0005.

Mentions: To better confirm whether changes in mitochondrial morphology are required for EMT, we assessed cell length and F-actin rearrangement in ARPE-19 cells under conditions in which mitochondrial fission or fusion were selectively suppressed by transfection with Drp1 or Mfn1 siRNAs. As shown in Figure 5A and B, inhibition of mitochondrial fusion by Mfn1 siRNA significantly blocked the increase in cell length induced by TGF-β compared to control ARPE-19 cells. Like OPA3 knockdown, inhibition of mitochondrial fission by Drp1 siRNA effectively increased cell length and induced F-actin rearrangement (Fig. 5). Consistent with this, the migration ability of the cells transfected with Drp1 siRNA was significantly increased after treatment of TGF-β (Fig. 5C). Inhibition of mitochondrial fusion by Mfn1 siRNA attenuated the TGF-β-induced cell migration (Fig. 5C). These results collectively suggest that proteins involved in mitochondrial elongation may play a role in TGF-β-induced EMT.


Downregulation of OPA3 is responsible for transforming growth factor-β-induced mitochondrial elongation and F-actin rearrangement in retinal pigment epithelial ARPE-19 cells.

Ryu SW, Yoon J, Yim N, Choi K, Choi C - PLoS ONE (2013)

Functional changes in cells induced by knockdown of proteins involved in mitochondrial dynamics.(A and B) The mitochondrial fusion by Drp1 knockdown leads to an increase of cell length and F-actin. APRE-19 cells were transfected with Drp1 siRNA, Mfn1 siRNA, or control siRNA. Forty-eight hours after transfection, cells were treated withTGF-β for 48 h. Cells were fixed and stained with phalloidin-TRITC (A). Cell length was analyzed using confocal images (B). Data are the mean ± SD of three experiments, each with 100 cells per condition. (C) The mitochondrial fusion by Drp1 knockdown promotes cell migration induced by TGF-β. Cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were treated with TGF-β for the indicated periods of time. The migrated cells were counted. Data shown represent the average of three independent experiments. *P<0.05; ***P<0.0005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643898&req=5

pone-0063495-g005: Functional changes in cells induced by knockdown of proteins involved in mitochondrial dynamics.(A and B) The mitochondrial fusion by Drp1 knockdown leads to an increase of cell length and F-actin. APRE-19 cells were transfected with Drp1 siRNA, Mfn1 siRNA, or control siRNA. Forty-eight hours after transfection, cells were treated withTGF-β for 48 h. Cells were fixed and stained with phalloidin-TRITC (A). Cell length was analyzed using confocal images (B). Data are the mean ± SD of three experiments, each with 100 cells per condition. (C) The mitochondrial fusion by Drp1 knockdown promotes cell migration induced by TGF-β. Cells were transfected with the indicated siRNAs. Forty-eight hours after transfection, cells were treated with TGF-β for the indicated periods of time. The migrated cells were counted. Data shown represent the average of three independent experiments. *P<0.05; ***P<0.0005.
Mentions: To better confirm whether changes in mitochondrial morphology are required for EMT, we assessed cell length and F-actin rearrangement in ARPE-19 cells under conditions in which mitochondrial fission or fusion were selectively suppressed by transfection with Drp1 or Mfn1 siRNAs. As shown in Figure 5A and B, inhibition of mitochondrial fusion by Mfn1 siRNA significantly blocked the increase in cell length induced by TGF-β compared to control ARPE-19 cells. Like OPA3 knockdown, inhibition of mitochondrial fission by Drp1 siRNA effectively increased cell length and induced F-actin rearrangement (Fig. 5). Consistent with this, the migration ability of the cells transfected with Drp1 siRNA was significantly increased after treatment of TGF-β (Fig. 5C). Inhibition of mitochondrial fusion by Mfn1 siRNA attenuated the TGF-β-induced cell migration (Fig. 5C). These results collectively suggest that proteins involved in mitochondrial elongation may play a role in TGF-β-induced EMT.

Bottom Line: We also showed that Drp1 depletion increased cell length and induced rearrangement of F-actin.Depletion of Mfn1 blocked the increase in cell length during TGF-β-mediated EMT.These results collectively substantiate the involvement of mitochondrial dynamics in TGF-β-induced EMT.

View Article: PubMed Central - PubMed

Affiliation: Department of Bio and Brain Engineering, KAIST, Daejeon, Korea. ryus@kaist.ac.kr

ABSTRACT
Transforming growth factor-β signaling is known to be a key signaling pathway in the induction of epithelial-mesenchymal transition. However, the mechanism of TGF-β signaling in the modulation of EMT remains unclear. In this study, we found that TGF-β treatment resulted in elongation of mitochondria accompanied by induction of N-cadherin, vimentin, and F-actin in retinal pigment epithelial cells. Moreover, OPA3, which plays a crucial role in mitochondrial dynamics, was downregulated following TGF-β treatment. Suppression of TGF-β signaling using Smad2 siRNA prevented loss of OPA3 induced by TGF-β. Knockdown of OPA3 by siRNA and inducible shRNA significantly increased stress fiber levels, cell length, cell migration and mitochondrial elongation. In contrast, forced expression of OPA3 in ARPE-19 cells inhibited F-actin rearrangement and induced mitochondrial fragmentation. We also showed that Drp1 depletion increased cell length and induced rearrangement of F-actin. Depletion of Mfn1 blocked the increase in cell length during TGF-β-mediated EMT. These results collectively substantiate the involvement of mitochondrial dynamics in TGF-β-induced EMT.

Show MeSH
Related in: MedlinePlus