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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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Knockdown of OPA3 induced changes in cell morphology and sensitized cells to F-actin rearrangement induced by TGF-β.(A and B) Effects of Smad2 on changes in cell morphology and OPA3 level by TGF-β treatment. (B) APRE-19 cells were transfected with Smad2 siRNA or control siRNA. Forty-eight hours after transfection, cells were incubated in the absence or presence of TGF-β for 48 h. Cells were analyzed with phase contrast microscopy (A). Real-time PCR evaluated the level of OPA3 mRNA (B). (C–G) Effects of OPA3 depletion on cell morphology and F-actin rearrangement. APRE-19 cells were transfected with OPA3 siRNA or control siRNA. Forty-eight hours after transfection, cells were treated with TGF-β for the indicated periods of time. Cells were analyzed by Western blotting (C) and Real-time PCR (D). Cells were fixed and stained with phalloidin-TRITC (E). TRITC intensity (F) and Cell lengths (G) were analyzed using confocal images. Data are the mean ± SD of three experiments, each with >50 cells per condition. *P<0.05; **P<0.005.
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pone-0063495-g002: Knockdown of OPA3 induced changes in cell morphology and sensitized cells to F-actin rearrangement induced by TGF-β.(A and B) Effects of Smad2 on changes in cell morphology and OPA3 level by TGF-β treatment. (B) APRE-19 cells were transfected with Smad2 siRNA or control siRNA. Forty-eight hours after transfection, cells were incubated in the absence or presence of TGF-β for 48 h. Cells were analyzed with phase contrast microscopy (A). Real-time PCR evaluated the level of OPA3 mRNA (B). (C–G) Effects of OPA3 depletion on cell morphology and F-actin rearrangement. APRE-19 cells were transfected with OPA3 siRNA or control siRNA. Forty-eight hours after transfection, cells were treated with TGF-β for the indicated periods of time. Cells were analyzed by Western blotting (C) and Real-time PCR (D). Cells were fixed and stained with phalloidin-TRITC (E). TRITC intensity (F) and Cell lengths (G) were analyzed using confocal images. Data are the mean ± SD of three experiments, each with >50 cells per condition. *P<0.05; **P<0.005.

Mentions: Smads have been well characterized as direct targets of TGF-β/TGF-β receptor signaling [2], [12]. Consistent with this, knockdown of Smad2 prevented the TGF-β-induced morphological changes (Fig. 2A). We next investigated whether blocking TGF-β signaling by Smad2 knockdown also restored the expression level of OPA3. After transient transfection of ARPE-19 cells with Smad2 siRNA, the level of Smad2 was significantly reduced compared to cells transfected with negative control siRNA (Figure S2). The basal mRNA level of OPA3 was slightly increased in cells transfected with Smad2 siRNA and dramatically reduced in control cells after TGF-β treatment compared to the control cells (Fig. 2B). Moreover, knockdown of Smad2 significantly blocked the reduction in OPA3 levels following TGF-β treatment (Fig. 2B). Consistent results were obtained for protein level of OPA3 (Figure S2). These data collectively indicate that Smad2 signaling is essential for TGF-β-induced reduction of OPA3 expression and possibly mitochondrial changes.


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)

Knockdown of OPA3 induced changes in cell morphology and sensitized cells to F-actin rearrangement induced by TGF-β.(A and B) Effects of Smad2 on changes in cell morphology and OPA3 level by TGF-β treatment. (B) APRE-19 cells were transfected with Smad2 siRNA or control siRNA. Forty-eight hours after transfection, cells were incubated in the absence or presence of TGF-β for 48 h. Cells were analyzed with phase contrast microscopy (A). Real-time PCR evaluated the level of OPA3 mRNA (B). (C–G) Effects of OPA3 depletion on cell morphology and F-actin rearrangement. APRE-19 cells were transfected with OPA3 siRNA or control siRNA. Forty-eight hours after transfection, cells were treated with TGF-β for the indicated periods of time. Cells were analyzed by Western blotting (C) and Real-time PCR (D). Cells were fixed and stained with phalloidin-TRITC (E). TRITC intensity (F) and Cell lengths (G) were analyzed using confocal images. Data are the mean ± SD of three experiments, each with >50 cells per condition. *P<0.05; **P<0.005.
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Related In: Results  -  Collection

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

pone-0063495-g002: Knockdown of OPA3 induced changes in cell morphology and sensitized cells to F-actin rearrangement induced by TGF-β.(A and B) Effects of Smad2 on changes in cell morphology and OPA3 level by TGF-β treatment. (B) APRE-19 cells were transfected with Smad2 siRNA or control siRNA. Forty-eight hours after transfection, cells were incubated in the absence or presence of TGF-β for 48 h. Cells were analyzed with phase contrast microscopy (A). Real-time PCR evaluated the level of OPA3 mRNA (B). (C–G) Effects of OPA3 depletion on cell morphology and F-actin rearrangement. APRE-19 cells were transfected with OPA3 siRNA or control siRNA. Forty-eight hours after transfection, cells were treated with TGF-β for the indicated periods of time. Cells were analyzed by Western blotting (C) and Real-time PCR (D). Cells were fixed and stained with phalloidin-TRITC (E). TRITC intensity (F) and Cell lengths (G) were analyzed using confocal images. Data are the mean ± SD of three experiments, each with >50 cells per condition. *P<0.05; **P<0.005.
Mentions: Smads have been well characterized as direct targets of TGF-β/TGF-β receptor signaling [2], [12]. Consistent with this, knockdown of Smad2 prevented the TGF-β-induced morphological changes (Fig. 2A). We next investigated whether blocking TGF-β signaling by Smad2 knockdown also restored the expression level of OPA3. After transient transfection of ARPE-19 cells with Smad2 siRNA, the level of Smad2 was significantly reduced compared to cells transfected with negative control siRNA (Figure S2). The basal mRNA level of OPA3 was slightly increased in cells transfected with Smad2 siRNA and dramatically reduced in control cells after TGF-β treatment compared to the control cells (Fig. 2B). Moreover, knockdown of Smad2 significantly blocked the reduction in OPA3 levels following TGF-β treatment (Fig. 2B). Consistent results were obtained for protein level of OPA3 (Figure S2). These data collectively indicate that Smad2 signaling is essential for TGF-β-induced reduction of OPA3 expression and possibly mitochondrial changes.

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