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Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT.

Vicidomini R, Di Giovanni A, Petrizzo A, Iannucci LF, Benvenuto G, Nagel AC, Preiss A, Furia M - Cell Death Dis (2015)

Bottom Line: Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells.Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted.On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia, Università di Napoli 'Federico II', via Cinthia, Naples 80126, Italy.

ABSTRACT
Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called 'undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell-cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

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Loss of Arm/β-catenin apical localization and reduced F-actin accumulation in the silenced discs. Confocal analysis of wing discs collected at 120 h AED and stained with Arm antibody (in red or gray) or phalloidin (red or gray). (a) Localization of Arm protein in en>GFP control discs, in which GFP (green) marks the en posterior domain. Note that Arm is strongly concentrated in two stripes of cells adjacent to the D/V boundary, and Z-stack analysis (right panels) shows its uniform apical localization in both A/P compartments. (b) Localization of Arm in an en>IRmfl silenced disc (v46282 line). mfl silencing causes a strong reduction of Arm protein, with Z-stack analysis (right panels) demonstrating loss of apical localization (right panel). In a and b, green dots mark the boundary between the A and P compartments within the enlargements; red dots mark XZ projection enlargements. (c and d) Confocal analysis of wing discs stained with phalloidin. (c) In en>GFP control discs, F-actin concentrates in two stripes of cells adjacent to the D/V boundary. (d) In en>GFP, en>IRmfl silenced discs (v46282 line in the picture), F-actin accumulation is strongly reduced within the whole silenced domain, and particularly at the D/V margin. GFP is in green and phalloidin in red or gray. Green dots mark the A/P boundary. a, Apical; b, basal
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fig6: Loss of Arm/β-catenin apical localization and reduced F-actin accumulation in the silenced discs. Confocal analysis of wing discs collected at 120 h AED and stained with Arm antibody (in red or gray) or phalloidin (red or gray). (a) Localization of Arm protein in en>GFP control discs, in which GFP (green) marks the en posterior domain. Note that Arm is strongly concentrated in two stripes of cells adjacent to the D/V boundary, and Z-stack analysis (right panels) shows its uniform apical localization in both A/P compartments. (b) Localization of Arm in an en>IRmfl silenced disc (v46282 line). mfl silencing causes a strong reduction of Arm protein, with Z-stack analysis (right panels) demonstrating loss of apical localization (right panel). In a and b, green dots mark the boundary between the A and P compartments within the enlargements; red dots mark XZ projection enlargements. (c and d) Confocal analysis of wing discs stained with phalloidin. (c) In en>GFP control discs, F-actin concentrates in two stripes of cells adjacent to the D/V boundary. (d) In en>GFP, en>IRmfl silenced discs (v46282 line in the picture), F-actin accumulation is strongly reduced within the whole silenced domain, and particularly at the D/V margin. GFP is in green and phalloidin in red or gray. Green dots mark the A/P boundary. a, Apical; b, basal

Mentions: The Arm protein is a known effector of Wg signaling and has a dual role: as a component of the cell adherens junctions on the one hand and as a nuclear transcription factor transducing Wg signal on the other hand.35 Within the wing disc, Arm concentrates apically, in which it binds transmembrane cadherins to build up the adherens junctions that connect actin filaments across polarized epithelial cells36 (see Figure 6a). Interestingly, upon mfl silencing directed by en-Gal4, Arm accumulation was strongly reduced in the whole silenced area, and most evidently at the D/V boundary. Moreover, confocal Z-stack analysis revealed a reduction of apical Arm accumulation, so that cells had lost their polarity (Figure 6b). Identical results where obtained when silencing was directed by the omb-Gal4 driver (Supplementary Figure 4). Reduction of apical Arm is consistent with the observation that Wg overexpression in the wing discs induces a transient reduction of membrane-associated Arm, this way allowing an immediate decline of cell adhesion that facilitates structural reorganization of the cytoskeleton.37 Similarly to Arm, F-actin accumulation was also heavily reduced, and accumulation at the D/V boundary disrupted (Figures 6c and d and Supplementary Videos 2 and 3). Hence, the concomitant induction of cell death and proliferation is accompanied by extensive cytoskeletal remodeling. The involvement of pseudouridine synthase activity in cytoskeletal dynamics and cell adhesion is worth noting, and it has previously been described also in human cells.38


Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT.

Vicidomini R, Di Giovanni A, Petrizzo A, Iannucci LF, Benvenuto G, Nagel AC, Preiss A, Furia M - Cell Death Dis (2015)

Loss of Arm/β-catenin apical localization and reduced F-actin accumulation in the silenced discs. Confocal analysis of wing discs collected at 120 h AED and stained with Arm antibody (in red or gray) or phalloidin (red or gray). (a) Localization of Arm protein in en>GFP control discs, in which GFP (green) marks the en posterior domain. Note that Arm is strongly concentrated in two stripes of cells adjacent to the D/V boundary, and Z-stack analysis (right panels) shows its uniform apical localization in both A/P compartments. (b) Localization of Arm in an en>IRmfl silenced disc (v46282 line). mfl silencing causes a strong reduction of Arm protein, with Z-stack analysis (right panels) demonstrating loss of apical localization (right panel). In a and b, green dots mark the boundary between the A and P compartments within the enlargements; red dots mark XZ projection enlargements. (c and d) Confocal analysis of wing discs stained with phalloidin. (c) In en>GFP control discs, F-actin concentrates in two stripes of cells adjacent to the D/V boundary. (d) In en>GFP, en>IRmfl silenced discs (v46282 line in the picture), F-actin accumulation is strongly reduced within the whole silenced domain, and particularly at the D/V margin. GFP is in green and phalloidin in red or gray. Green dots mark the A/P boundary. a, Apical; b, basal
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fig6: Loss of Arm/β-catenin apical localization and reduced F-actin accumulation in the silenced discs. Confocal analysis of wing discs collected at 120 h AED and stained with Arm antibody (in red or gray) or phalloidin (red or gray). (a) Localization of Arm protein in en>GFP control discs, in which GFP (green) marks the en posterior domain. Note that Arm is strongly concentrated in two stripes of cells adjacent to the D/V boundary, and Z-stack analysis (right panels) shows its uniform apical localization in both A/P compartments. (b) Localization of Arm in an en>IRmfl silenced disc (v46282 line). mfl silencing causes a strong reduction of Arm protein, with Z-stack analysis (right panels) demonstrating loss of apical localization (right panel). In a and b, green dots mark the boundary between the A and P compartments within the enlargements; red dots mark XZ projection enlargements. (c and d) Confocal analysis of wing discs stained with phalloidin. (c) In en>GFP control discs, F-actin concentrates in two stripes of cells adjacent to the D/V boundary. (d) In en>GFP, en>IRmfl silenced discs (v46282 line in the picture), F-actin accumulation is strongly reduced within the whole silenced domain, and particularly at the D/V margin. GFP is in green and phalloidin in red or gray. Green dots mark the A/P boundary. a, Apical; b, basal
Mentions: The Arm protein is a known effector of Wg signaling and has a dual role: as a component of the cell adherens junctions on the one hand and as a nuclear transcription factor transducing Wg signal on the other hand.35 Within the wing disc, Arm concentrates apically, in which it binds transmembrane cadherins to build up the adherens junctions that connect actin filaments across polarized epithelial cells36 (see Figure 6a). Interestingly, upon mfl silencing directed by en-Gal4, Arm accumulation was strongly reduced in the whole silenced area, and most evidently at the D/V boundary. Moreover, confocal Z-stack analysis revealed a reduction of apical Arm accumulation, so that cells had lost their polarity (Figure 6b). Identical results where obtained when silencing was directed by the omb-Gal4 driver (Supplementary Figure 4). Reduction of apical Arm is consistent with the observation that Wg overexpression in the wing discs induces a transient reduction of membrane-associated Arm, this way allowing an immediate decline of cell adhesion that facilitates structural reorganization of the cytoskeleton.37 Similarly to Arm, F-actin accumulation was also heavily reduced, and accumulation at the D/V boundary disrupted (Figures 6c and d and Supplementary Videos 2 and 3). Hence, the concomitant induction of cell death and proliferation is accompanied by extensive cytoskeletal remodeling. The involvement of pseudouridine synthase activity in cytoskeletal dynamics and cell adhesion is worth noting, and it has previously been described also in human cells.38

Bottom Line: Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells.Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted.On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia, Università di Napoli 'Federico II', via Cinthia, Naples 80126, Italy.

ABSTRACT
Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called 'undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell-cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

Show MeSH
Related in: MedlinePlus