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Pelle Modulates dFoxO-Mediated Cell Death in Drosophila.

Wu C, Chen Y, Wang F, Chen C, Zhang S, Li C, Li W, Wu S, Xue L - PLoS Genet. (2015)

Bottom Line: Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals.Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly.This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Interventional Radiology, Shanghai 10th People's Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, Shanghai, China.

ABSTRACT
Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. Here we report a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. We found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Loss of pll promotes dFoxO nuclear localization and transcriptional activity.(A) Loss of pll does not affect the transcription of dFoxO. Histogram showing the levels of dFoxO mRNAs measured by quantitative RT-PCR. Total RNA of Drosophila third instar larvae were extracted and normalized for cDNA synthesis. Error bars represents standard deviation from three independent experiments. ns stands for not significant. (B-D) Loss of pll promotes nuclear localization of dFoxO. (B) Quantification of the nuclear/cytoplasmic ratio of dFoxO-GFP fusion protein in the fat body shown in C and D. dFoxO-GFP intensities were measured in pixels using Image J. Error bars showed standard deviation from measurement of at least 15 cells for each genotype. Unpaired t test was used to calculate statistical significance, indicated with asterisks (*** P<0.001). (C and D) Fluorescence micrographs of fat body cells are shown. Compared with the control (C), loss of pll promotes the translocation of dFoxO-GFP from cytoplasm to nucleus (D). Nuclei were marked with DAPI (blue), cell membranes were stained by anti-Dlg antibody (red). (E and F) X-Gal staining of a Thor-LacZ reporter in third instar larval wing discs. Compared with the control (E), knock-down pll in the wing pouch induced Thor transcription (F). (G) Loss of pll up-regulated the level of Thor mRNA, as measured by quantitative RT-PCR. Total RNA of Drosophila third instar larvae were extracted and normalized for cDNA synthesis. Error bars represented standard deviation from three independent experiments. Unpaired t test was used to calculate statistical significance, indicated with asterisks (* P<0.05). Detailed genotypes: (A) Left: act-Gal4/+, Right: UAS-pll-IRV2889/+; act-Gal4/+ (B) Left: Cg-Gal4/+, Right: Cg-Gal4/UAS-pll-IRV2889 (C) Cg-Gal4/+; dFoxO-GFP/+ (D) Cg-Gal4/UAS-pll-IRV2889; dFoxO-GFP/+ (E) Sd-Gal4/+; Thor-LacZ/+ (F) Sd-Gal4/+; Thor-LacZ/UAS-pll-IRV2889 (G) Left: act-Gal4/+, Right: UAS-pll-IRV2889/+; act-Gal4/+.
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pgen.1005589.g009: Loss of pll promotes dFoxO nuclear localization and transcriptional activity.(A) Loss of pll does not affect the transcription of dFoxO. Histogram showing the levels of dFoxO mRNAs measured by quantitative RT-PCR. Total RNA of Drosophila third instar larvae were extracted and normalized for cDNA synthesis. Error bars represents standard deviation from three independent experiments. ns stands for not significant. (B-D) Loss of pll promotes nuclear localization of dFoxO. (B) Quantification of the nuclear/cytoplasmic ratio of dFoxO-GFP fusion protein in the fat body shown in C and D. dFoxO-GFP intensities were measured in pixels using Image J. Error bars showed standard deviation from measurement of at least 15 cells for each genotype. Unpaired t test was used to calculate statistical significance, indicated with asterisks (*** P<0.001). (C and D) Fluorescence micrographs of fat body cells are shown. Compared with the control (C), loss of pll promotes the translocation of dFoxO-GFP from cytoplasm to nucleus (D). Nuclei were marked with DAPI (blue), cell membranes were stained by anti-Dlg antibody (red). (E and F) X-Gal staining of a Thor-LacZ reporter in third instar larval wing discs. Compared with the control (E), knock-down pll in the wing pouch induced Thor transcription (F). (G) Loss of pll up-regulated the level of Thor mRNA, as measured by quantitative RT-PCR. Total RNA of Drosophila third instar larvae were extracted and normalized for cDNA synthesis. Error bars represented standard deviation from three independent experiments. Unpaired t test was used to calculate statistical significance, indicated with asterisks (* P<0.05). Detailed genotypes: (A) Left: act-Gal4/+, Right: UAS-pll-IRV2889/+; act-Gal4/+ (B) Left: Cg-Gal4/+, Right: Cg-Gal4/UAS-pll-IRV2889 (C) Cg-Gal4/+; dFoxO-GFP/+ (D) Cg-Gal4/UAS-pll-IRV2889; dFoxO-GFP/+ (E) Sd-Gal4/+; Thor-LacZ/+ (F) Sd-Gal4/+; Thor-LacZ/UAS-pll-IRV2889 (G) Left: act-Gal4/+, Right: UAS-pll-IRV2889/+; act-Gal4/+.

Mentions: To address how Pll modulates dFoxO activity, we first checked whether Pll regulates dFoxO transcription by executing a real-time qRT-PCR assay. We found that a ubiquitous knockdown of pll (act>pll-IR) did not affect the mRNA level of dFoxO, as compared to the act-Gal4 control (Fig 9A), suggesting that pll does not regulate dFoxO transcription. Previous studies indicate that the nuclear localization of dFoxO is regulated by a series of post-translational modifications, including phosphorylation by different kinases [34]. To examine whether Pll modulates the nuclear-cytoplasmic shuttling of dFoxO, we checked the sub-cellular localization of a dFoxO-GFP fusion protein in the fat body. We found that the nuclear localization of dFoxO-GFP was significantly increased when pll was knocked down in fat body cells by Cg-Gal4 (Fig 9B–9D), which drives Gal4 expression specifically in fat body and hemocytes under the control of collagen (Cg25C) promoter [47]. To monitor the dFoxO activity directly, we detected the expression of its well-characterized target gene Thor/4E-BP by a Thor-LacZ reporter [26,28,48,49]. We found that Thor-LacZ expression was distinctly induced in the wing pouch by RNAi inactivation of pll under the control of Sd-Gal4 (Fig 9E and 9F). In addition, qRT-PCR assay confirmed that depletion of pll resulted in up-regulated Thor/4E-BP transcription (Fig 9G). Thus, we conclude that Pll regulates dFoxO subcellular localization and transcriptional activity.


Pelle Modulates dFoxO-Mediated Cell Death in Drosophila.

Wu C, Chen Y, Wang F, Chen C, Zhang S, Li C, Li W, Wu S, Xue L - PLoS Genet. (2015)

Loss of pll promotes dFoxO nuclear localization and transcriptional activity.(A) Loss of pll does not affect the transcription of dFoxO. Histogram showing the levels of dFoxO mRNAs measured by quantitative RT-PCR. Total RNA of Drosophila third instar larvae were extracted and normalized for cDNA synthesis. Error bars represents standard deviation from three independent experiments. ns stands for not significant. (B-D) Loss of pll promotes nuclear localization of dFoxO. (B) Quantification of the nuclear/cytoplasmic ratio of dFoxO-GFP fusion protein in the fat body shown in C and D. dFoxO-GFP intensities were measured in pixels using Image J. Error bars showed standard deviation from measurement of at least 15 cells for each genotype. Unpaired t test was used to calculate statistical significance, indicated with asterisks (*** P<0.001). (C and D) Fluorescence micrographs of fat body cells are shown. Compared with the control (C), loss of pll promotes the translocation of dFoxO-GFP from cytoplasm to nucleus (D). Nuclei were marked with DAPI (blue), cell membranes were stained by anti-Dlg antibody (red). (E and F) X-Gal staining of a Thor-LacZ reporter in third instar larval wing discs. Compared with the control (E), knock-down pll in the wing pouch induced Thor transcription (F). (G) Loss of pll up-regulated the level of Thor mRNA, as measured by quantitative RT-PCR. Total RNA of Drosophila third instar larvae were extracted and normalized for cDNA synthesis. Error bars represented standard deviation from three independent experiments. Unpaired t test was used to calculate statistical significance, indicated with asterisks (* P<0.05). Detailed genotypes: (A) Left: act-Gal4/+, Right: UAS-pll-IRV2889/+; act-Gal4/+ (B) Left: Cg-Gal4/+, Right: Cg-Gal4/UAS-pll-IRV2889 (C) Cg-Gal4/+; dFoxO-GFP/+ (D) Cg-Gal4/UAS-pll-IRV2889; dFoxO-GFP/+ (E) Sd-Gal4/+; Thor-LacZ/+ (F) Sd-Gal4/+; Thor-LacZ/UAS-pll-IRV2889 (G) Left: act-Gal4/+, Right: UAS-pll-IRV2889/+; act-Gal4/+.
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pgen.1005589.g009: Loss of pll promotes dFoxO nuclear localization and transcriptional activity.(A) Loss of pll does not affect the transcription of dFoxO. Histogram showing the levels of dFoxO mRNAs measured by quantitative RT-PCR. Total RNA of Drosophila third instar larvae were extracted and normalized for cDNA synthesis. Error bars represents standard deviation from three independent experiments. ns stands for not significant. (B-D) Loss of pll promotes nuclear localization of dFoxO. (B) Quantification of the nuclear/cytoplasmic ratio of dFoxO-GFP fusion protein in the fat body shown in C and D. dFoxO-GFP intensities were measured in pixels using Image J. Error bars showed standard deviation from measurement of at least 15 cells for each genotype. Unpaired t test was used to calculate statistical significance, indicated with asterisks (*** P<0.001). (C and D) Fluorescence micrographs of fat body cells are shown. Compared with the control (C), loss of pll promotes the translocation of dFoxO-GFP from cytoplasm to nucleus (D). Nuclei were marked with DAPI (blue), cell membranes were stained by anti-Dlg antibody (red). (E and F) X-Gal staining of a Thor-LacZ reporter in third instar larval wing discs. Compared with the control (E), knock-down pll in the wing pouch induced Thor transcription (F). (G) Loss of pll up-regulated the level of Thor mRNA, as measured by quantitative RT-PCR. Total RNA of Drosophila third instar larvae were extracted and normalized for cDNA synthesis. Error bars represented standard deviation from three independent experiments. Unpaired t test was used to calculate statistical significance, indicated with asterisks (* P<0.05). Detailed genotypes: (A) Left: act-Gal4/+, Right: UAS-pll-IRV2889/+; act-Gal4/+ (B) Left: Cg-Gal4/+, Right: Cg-Gal4/UAS-pll-IRV2889 (C) Cg-Gal4/+; dFoxO-GFP/+ (D) Cg-Gal4/UAS-pll-IRV2889; dFoxO-GFP/+ (E) Sd-Gal4/+; Thor-LacZ/+ (F) Sd-Gal4/+; Thor-LacZ/UAS-pll-IRV2889 (G) Left: act-Gal4/+, Right: UAS-pll-IRV2889/+; act-Gal4/+.
Mentions: To address how Pll modulates dFoxO activity, we first checked whether Pll regulates dFoxO transcription by executing a real-time qRT-PCR assay. We found that a ubiquitous knockdown of pll (act>pll-IR) did not affect the mRNA level of dFoxO, as compared to the act-Gal4 control (Fig 9A), suggesting that pll does not regulate dFoxO transcription. Previous studies indicate that the nuclear localization of dFoxO is regulated by a series of post-translational modifications, including phosphorylation by different kinases [34]. To examine whether Pll modulates the nuclear-cytoplasmic shuttling of dFoxO, we checked the sub-cellular localization of a dFoxO-GFP fusion protein in the fat body. We found that the nuclear localization of dFoxO-GFP was significantly increased when pll was knocked down in fat body cells by Cg-Gal4 (Fig 9B–9D), which drives Gal4 expression specifically in fat body and hemocytes under the control of collagen (Cg25C) promoter [47]. To monitor the dFoxO activity directly, we detected the expression of its well-characterized target gene Thor/4E-BP by a Thor-LacZ reporter [26,28,48,49]. We found that Thor-LacZ expression was distinctly induced in the wing pouch by RNAi inactivation of pll under the control of Sd-Gal4 (Fig 9E and 9F). In addition, qRT-PCR assay confirmed that depletion of pll resulted in up-regulated Thor/4E-BP transcription (Fig 9G). Thus, we conclude that Pll regulates dFoxO subcellular localization and transcriptional activity.

Bottom Line: Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals.Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly.This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Interventional Radiology, Shanghai 10th People's Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, Shanghai, China.

ABSTRACT
Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. Here we report a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. We found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

No MeSH data available.


Related in: MedlinePlus