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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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Mfl depletion induces JNK and Mmp1 ectopic activation. Confocal analysis of wing discs collected at 120 h AED. (a) In omb>puc-lacZ control discs, activation of the JNK pathway, marked by the puc-lacZ reporter, is restricted to the stalk cells (inset) and to some dispersed cells.47 (b) In omb>IRmfl; puc-lacZ silenced discs (v46282 line in the picture), JNK is ectopically and strongly activated. In (a and b), DAPI (4',6-diamidino-2-phenylindole) is in blue, Mfl in green and β-gal in red. (c) Mmp1 expression in omb>GFP control discs marks the trachea (arrow) and the stalk cells (not in frame), where it overlaps puc-lacZ expression. Z-stack analysis (see XZ and YZ projections) confirms Mmp1 absence in the omb domain. (d) In omb>IRmfl silenced discs (v46282 line in the picture), Mmp1is strongly and ectopically induced in the MFL-depleted domain; in the D compartment, Mmp1 enrichment flanks the A/P border (closed arrowhead) within the area of JNK activation (compared with b), whereas in the V compartment, Mmp1 concentrates in a small domain (open arrowhead) overlapping the Wg-expressing inner ring. In both areas, Z-stack analysis (see XZ and YZ projections) confirms Mmp1 active secretion within the silenced domain. DAPI is in blue; GFP (c) and Mfl (d) in green and Mmp1 in red. a, Apical; b, basal
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fig7: Mfl depletion induces JNK and Mmp1 ectopic activation. Confocal analysis of wing discs collected at 120 h AED. (a) In omb>puc-lacZ control discs, activation of the JNK pathway, marked by the puc-lacZ reporter, is restricted to the stalk cells (inset) and to some dispersed cells.47 (b) In omb>IRmfl; puc-lacZ silenced discs (v46282 line in the picture), JNK is ectopically and strongly activated. In (a and b), DAPI (4',6-diamidino-2-phenylindole) is in blue, Mfl in green and β-gal in red. (c) Mmp1 expression in omb>GFP control discs marks the trachea (arrow) and the stalk cells (not in frame), where it overlaps puc-lacZ expression. Z-stack analysis (see XZ and YZ projections) confirms Mmp1 absence in the omb domain. (d) In omb>IRmfl silenced discs (v46282 line in the picture), Mmp1is strongly and ectopically induced in the MFL-depleted domain; in the D compartment, Mmp1 enrichment flanks the A/P border (closed arrowhead) within the area of JNK activation (compared with b), whereas in the V compartment, Mmp1 concentrates in a small domain (open arrowhead) overlapping the Wg-expressing inner ring. In both areas, Z-stack analysis (see XZ and YZ projections) confirms Mmp1 active secretion within the silenced domain. DAPI is in blue; GFP (c) and Mfl (d) in green and Mmp1 in red. a, Apical; b, basal

Mentions: Given that the JNK pathway is known to be involved in cytoskeletal remodeling during both apoptotic39, 40 and regenerative processes,41, 42, 43, 44, 45 we checked whether it was specifically induced upon Mfl depletion. We then followed the expression of puckered (puc), a JNK downstream effector,46 taking advantage of the widely used puc-lacz reporter. As described,47puc-lacz expression in wild-type discs is restricted to the stalk region, where wing discs are connected to the larval epidermis (Figure 7a). In contrast, expression of this reporter was found strongly induced within the silenced discs (Figure 7b). Along the A/P border, JNK ectopic induction matched the local clusters of pyknotic nuclei, suggesting that it resulted in a local cell death. However, in the ventral regions it overlapped the areas of Wg accumulation, suggesting that in these regions JNK activity could instead promote proliferation, in keeping with the dual role recently suggested for this pathway.45 JNK also has a well conserved role in the induction of Mmps that degrade the extracellular matrix and are strongly expressed during regeneration.48, 49 Specifically, Drosophila Mmp1 is directly involved in re-epithelialization after wound healing, remodeling of the basement membrane and cytoskeletal reorganization.50, 51 Not surprisingly, Mmp1 was strongly induced along the A/P border and ventrally, where it matched the area of Wg overexpression at the middle of the inner ring (Figures 7c and d).


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)

Mfl depletion induces JNK and Mmp1 ectopic activation. Confocal analysis of wing discs collected at 120 h AED. (a) In omb>puc-lacZ control discs, activation of the JNK pathway, marked by the puc-lacZ reporter, is restricted to the stalk cells (inset) and to some dispersed cells.47 (b) In omb>IRmfl; puc-lacZ silenced discs (v46282 line in the picture), JNK is ectopically and strongly activated. In (a and b), DAPI (4',6-diamidino-2-phenylindole) is in blue, Mfl in green and β-gal in red. (c) Mmp1 expression in omb>GFP control discs marks the trachea (arrow) and the stalk cells (not in frame), where it overlaps puc-lacZ expression. Z-stack analysis (see XZ and YZ projections) confirms Mmp1 absence in the omb domain. (d) In omb>IRmfl silenced discs (v46282 line in the picture), Mmp1is strongly and ectopically induced in the MFL-depleted domain; in the D compartment, Mmp1 enrichment flanks the A/P border (closed arrowhead) within the area of JNK activation (compared with b), whereas in the V compartment, Mmp1 concentrates in a small domain (open arrowhead) overlapping the Wg-expressing inner ring. In both areas, Z-stack analysis (see XZ and YZ projections) confirms Mmp1 active secretion within the silenced domain. DAPI is in blue; GFP (c) and Mfl (d) in green and Mmp1 in red. a, Apical; b, basal
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fig7: Mfl depletion induces JNK and Mmp1 ectopic activation. Confocal analysis of wing discs collected at 120 h AED. (a) In omb>puc-lacZ control discs, activation of the JNK pathway, marked by the puc-lacZ reporter, is restricted to the stalk cells (inset) and to some dispersed cells.47 (b) In omb>IRmfl; puc-lacZ silenced discs (v46282 line in the picture), JNK is ectopically and strongly activated. In (a and b), DAPI (4',6-diamidino-2-phenylindole) is in blue, Mfl in green and β-gal in red. (c) Mmp1 expression in omb>GFP control discs marks the trachea (arrow) and the stalk cells (not in frame), where it overlaps puc-lacZ expression. Z-stack analysis (see XZ and YZ projections) confirms Mmp1 absence in the omb domain. (d) In omb>IRmfl silenced discs (v46282 line in the picture), Mmp1is strongly and ectopically induced in the MFL-depleted domain; in the D compartment, Mmp1 enrichment flanks the A/P border (closed arrowhead) within the area of JNK activation (compared with b), whereas in the V compartment, Mmp1 concentrates in a small domain (open arrowhead) overlapping the Wg-expressing inner ring. In both areas, Z-stack analysis (see XZ and YZ projections) confirms Mmp1 active secretion within the silenced domain. DAPI is in blue; GFP (c) and Mfl (d) in green and Mmp1 in red. a, Apical; b, basal
Mentions: Given that the JNK pathway is known to be involved in cytoskeletal remodeling during both apoptotic39, 40 and regenerative processes,41, 42, 43, 44, 45 we checked whether it was specifically induced upon Mfl depletion. We then followed the expression of puckered (puc), a JNK downstream effector,46 taking advantage of the widely used puc-lacz reporter. As described,47puc-lacz expression in wild-type discs is restricted to the stalk region, where wing discs are connected to the larval epidermis (Figure 7a). In contrast, expression of this reporter was found strongly induced within the silenced discs (Figure 7b). Along the A/P border, JNK ectopic induction matched the local clusters of pyknotic nuclei, suggesting that it resulted in a local cell death. However, in the ventral regions it overlapped the areas of Wg accumulation, suggesting that in these regions JNK activity could instead promote proliferation, in keeping with the dual role recently suggested for this pathway.45 JNK also has a well conserved role in the induction of Mmps that degrade the extracellular matrix and are strongly expressed during regeneration.48, 49 Specifically, Drosophila Mmp1 is directly involved in re-epithelialization after wound healing, remodeling of the basement membrane and cytoskeletal reorganization.50, 51 Not surprisingly, Mmp1 was strongly induced along the A/P border and ventrally, where it matched the area of Wg overexpression at the middle of the inner ring (Figures 7c and d).

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