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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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Stable knockdown of OPA3 induced the rearrangement of F-actin and mitochondrial elongation in HeLa cells.(A–D) Effect of OPA3 knockdown on changes in cell morphology. HeLa cells were transfected with an inducible OPA3 shRNA plasmid (Tet-OPA3) and then selected with G418 for 2 weeks. After selection, cells were incubated in the absence or presence of doxycycline (Dox) for 3 days. For Western blotting with the indicated antibodies (A), cells were harvested and then lysed. Cell morphology (B) and cell length (C) was analyzed using phase contrast images. Data are the mean ± SD of three experiments, each with 100 cells per condition. For confocal analysis (D), cells were fixed and stained with anti-Tom20 antibody (green) and phalloidin-TRITC (red). Higher magnification images of the highlighted areas are presented in the panels to the right. For quantification of mitochondrial fusion activity, live cells with mito-YFP were analyzed by photobleaching. Each line represents the mean of >30 measurements. ***P<0.0005.
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pone-0063495-g004: Stable knockdown of OPA3 induced the rearrangement of F-actin and mitochondrial elongation in HeLa cells.(A–D) Effect of OPA3 knockdown on changes in cell morphology. HeLa cells were transfected with an inducible OPA3 shRNA plasmid (Tet-OPA3) and then selected with G418 for 2 weeks. After selection, cells were incubated in the absence or presence of doxycycline (Dox) for 3 days. For Western blotting with the indicated antibodies (A), cells were harvested and then lysed. Cell morphology (B) and cell length (C) was analyzed using phase contrast images. Data are the mean ± SD of three experiments, each with 100 cells per condition. For confocal analysis (D), cells were fixed and stained with anti-Tom20 antibody (green) and phalloidin-TRITC (red). Higher magnification images of the highlighted areas are presented in the panels to the right. For quantification of mitochondrial fusion activity, live cells with mito-YFP were analyzed by photobleaching. Each line represents the mean of >30 measurements. ***P<0.0005.

Mentions: To confirm the effect of OPA3 in other cell types, we tested HeLa cells stably expressing doxycycline (Dox)-inducible OPA3 shRNA. The level of OPA3 protein in OPA3-shRNA cells was significantly reduced by Dox treatment (Fig. 4A). Consistent with previous results in ARPE-19 cells, the mesenchymal markers such as N-cadherin and cofilin were dramatically increased; while E-cadherin was reduced in inducible OPA3-shRNA cells after Dox treatment (Fig. 4A). We also observed the EMT-like morphological changes in OPA3-shRNA cells after Dox treatment (Fig. 4B and C). For analysis of F-actin rearrangement and mitochondrial morphology, inducible OPA3-shRNA cells were stained with phalloidin and an anti-Tom20 antibody. As shown in Figure 4D, weak F-actin staining was observed at the edges of cells before Dox treatment, whereas strong F-actin staining was detected throughout the cells after Dox treatment. Consistent with our previous report [10], depletion of OPA3 induced elongation of the mitochondrial network (Fig. 4D, green) and mitochondrial fusion activity (Fig. 4E).


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)

Stable knockdown of OPA3 induced the rearrangement of F-actin and mitochondrial elongation in HeLa cells.(A–D) Effect of OPA3 knockdown on changes in cell morphology. HeLa cells were transfected with an inducible OPA3 shRNA plasmid (Tet-OPA3) and then selected with G418 for 2 weeks. After selection, cells were incubated in the absence or presence of doxycycline (Dox) for 3 days. For Western blotting with the indicated antibodies (A), cells were harvested and then lysed. Cell morphology (B) and cell length (C) was analyzed using phase contrast images. Data are the mean ± SD of three experiments, each with 100 cells per condition. For confocal analysis (D), cells were fixed and stained with anti-Tom20 antibody (green) and phalloidin-TRITC (red). Higher magnification images of the highlighted areas are presented in the panels to the right. For quantification of mitochondrial fusion activity, live cells with mito-YFP were analyzed by photobleaching. Each line represents the mean of >30 measurements. ***P<0.0005.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643898&req=5

pone-0063495-g004: Stable knockdown of OPA3 induced the rearrangement of F-actin and mitochondrial elongation in HeLa cells.(A–D) Effect of OPA3 knockdown on changes in cell morphology. HeLa cells were transfected with an inducible OPA3 shRNA plasmid (Tet-OPA3) and then selected with G418 for 2 weeks. After selection, cells were incubated in the absence or presence of doxycycline (Dox) for 3 days. For Western blotting with the indicated antibodies (A), cells were harvested and then lysed. Cell morphology (B) and cell length (C) was analyzed using phase contrast images. Data are the mean ± SD of three experiments, each with 100 cells per condition. For confocal analysis (D), cells were fixed and stained with anti-Tom20 antibody (green) and phalloidin-TRITC (red). Higher magnification images of the highlighted areas are presented in the panels to the right. For quantification of mitochondrial fusion activity, live cells with mito-YFP were analyzed by photobleaching. Each line represents the mean of >30 measurements. ***P<0.0005.
Mentions: To confirm the effect of OPA3 in other cell types, we tested HeLa cells stably expressing doxycycline (Dox)-inducible OPA3 shRNA. The level of OPA3 protein in OPA3-shRNA cells was significantly reduced by Dox treatment (Fig. 4A). Consistent with previous results in ARPE-19 cells, the mesenchymal markers such as N-cadherin and cofilin were dramatically increased; while E-cadherin was reduced in inducible OPA3-shRNA cells after Dox treatment (Fig. 4A). We also observed the EMT-like morphological changes in OPA3-shRNA cells after Dox treatment (Fig. 4B and C). For analysis of F-actin rearrangement and mitochondrial morphology, inducible OPA3-shRNA cells were stained with phalloidin and an anti-Tom20 antibody. As shown in Figure 4D, weak F-actin staining was observed at the edges of cells before Dox treatment, whereas strong F-actin staining was detected throughout the cells after Dox treatment. Consistent with our previous report [10], depletion of OPA3 induced elongation of the mitochondrial network (Fig. 4D, green) and mitochondrial fusion activity (Fig. 4E).

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