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Uracil-induced signaling pathways for DUOX-dependent gut immunity.

Lee KA, Kim B, You H, Lee WJ - Fly (Austin) (2015)

Bottom Line: We have recently found that uracil stimulates Hedgehog signaling, which in turn upregulates cadherin99C (Cad99C) expression in enterocytes.These observations illustrate the complexity of signaling cascades in uracil-induced signaling pathways.Moreover, we will provide a brief discussion on two different models for uracil recognition and uracil-induced DUOX activation in Drosophila enterocytes.

View Article: PubMed Central - PubMed

Affiliation: a School of Biological Science, Seoul National University and National Creative Research Initiative Center for Hologenomics, Seoul National University , Seoul , South Korea.

ABSTRACT
Intestinal dual oxidase (DUOX) activation is the first line of host defense against enteric infection in Drosophila. DUOX enzymatic activity is mainly controlled by phospholipase C-β (PLCβ)-dependent calcium mobilization, whereas DUOX gene expression is mainly controlled by the MEKK1-p38 mitogen-activated protein kinase pathway. Furthermore, bacterial-derived uracil molecules act as ligands for DUOX activation. However, our current understanding of uracil-induced signal transduction pathways remain incomplete. We have recently found that uracil stimulates Hedgehog signaling, which in turn upregulates cadherin99C (Cad99C) expression in enterocytes. Cad99C molecules, along with PLCβ and protein kinase C, induce the formation of signaling endosomes that facilitate intracellular calcium mobilization for DUOX activity. These observations illustrate the complexity of signaling cascades in uracil-induced signaling pathways. Here, we further demonstrated the role of lipid raft formation and calmodulin-dependent protein kinase-II on endosome formation and calcium mobilization, respectively. Moreover, we will provide a brief discussion on two different models for uracil recognition and uracil-induced DUOX activation in Drosophila enterocytes.

No MeSH data available.


Related in: MedlinePlus

CaMKII acts as a downstream component of Hh-Cad99C, which required for DUOX activity. (A) Uracil-induced DUOX activation was abolished in CaMKII-KD flies. Uracil (20 nM) was administered orally to 3-day-old adult flies for 90 min, and DUOX-dependent ROS production in the midgut was visualized by HOCl−specific R19S dye (orange). Representative confocal microscopic images and the percentage of ROS-positive intestines is shown. (B) Epistatic analyses. Constitutive ROS generation found in flies carrying constitutive Hh signaling (Cos2-KD) can be abolished in the presence of CaMKII-KD. (C) CaMKII is required for uracil-induced Ca2+ mobilization. FRET analysis for the measurement of Ca2+ using flies expressing cameleon calcium sensor was performed. FRET efficiency (E %) was expressed as the mean ± SD from at least 30 flies. (D) Forced calcium increase is sufficient to induce DUOX activation in a CaMKII-KD animals. Flies over-expressing dominant-negative form of sarco/endoplasmic reticulum Ca2+-ATPase (SercaDN) were used. Data in A-D were analyzed using an ANOVA followed by Tukey post hoc test (C) or by Tamhane's T2 post hoc test (A,B and D); values represent mean ± SEM (*P < 0.05, **P < 0.005, ***P < 0.001) of at least three independent experiments.
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f0003: CaMKII acts as a downstream component of Hh-Cad99C, which required for DUOX activity. (A) Uracil-induced DUOX activation was abolished in CaMKII-KD flies. Uracil (20 nM) was administered orally to 3-day-old adult flies for 90 min, and DUOX-dependent ROS production in the midgut was visualized by HOCl−specific R19S dye (orange). Representative confocal microscopic images and the percentage of ROS-positive intestines is shown. (B) Epistatic analyses. Constitutive ROS generation found in flies carrying constitutive Hh signaling (Cos2-KD) can be abolished in the presence of CaMKII-KD. (C) CaMKII is required for uracil-induced Ca2+ mobilization. FRET analysis for the measurement of Ca2+ using flies expressing cameleon calcium sensor was performed. FRET efficiency (E %) was expressed as the mean ± SD from at least 30 flies. (D) Forced calcium increase is sufficient to induce DUOX activation in a CaMKII-KD animals. Flies over-expressing dominant-negative form of sarco/endoplasmic reticulum Ca2+-ATPase (SercaDN) were used. Data in A-D were analyzed using an ANOVA followed by Tukey post hoc test (C) or by Tamhane's T2 post hoc test (A,B and D); values represent mean ± SEM (*P < 0.05, **P < 0.005, ***P < 0.001) of at least three independent experiments.

Mentions: To determine whether the high infection-induced lethality observed in CaMKII-KD flies was due to impaired DUOX-dependent ROS generation, we measured uracil-induced ROS generation in these animals. We found that CaMKII-KD animals failed to generate ROS following uracil ingestion (Fig. 3A), indicating that CaMKII is required for DUOX activation. Epistatic analyses further revealed that constitutive ROS generation found in flies carrying constitutive Hh signaling could be abolished in the presence of CaMKII-KD (Fig. 3B). These results indicate that CaMKII acts as a downstream component of Hh signaling.Figure 3.


Uracil-induced signaling pathways for DUOX-dependent gut immunity.

Lee KA, Kim B, You H, Lee WJ - Fly (Austin) (2015)

CaMKII acts as a downstream component of Hh-Cad99C, which required for DUOX activity. (A) Uracil-induced DUOX activation was abolished in CaMKII-KD flies. Uracil (20 nM) was administered orally to 3-day-old adult flies for 90 min, and DUOX-dependent ROS production in the midgut was visualized by HOCl−specific R19S dye (orange). Representative confocal microscopic images and the percentage of ROS-positive intestines is shown. (B) Epistatic analyses. Constitutive ROS generation found in flies carrying constitutive Hh signaling (Cos2-KD) can be abolished in the presence of CaMKII-KD. (C) CaMKII is required for uracil-induced Ca2+ mobilization. FRET analysis for the measurement of Ca2+ using flies expressing cameleon calcium sensor was performed. FRET efficiency (E %) was expressed as the mean ± SD from at least 30 flies. (D) Forced calcium increase is sufficient to induce DUOX activation in a CaMKII-KD animals. Flies over-expressing dominant-negative form of sarco/endoplasmic reticulum Ca2+-ATPase (SercaDN) were used. Data in A-D were analyzed using an ANOVA followed by Tukey post hoc test (C) or by Tamhane's T2 post hoc test (A,B and D); values represent mean ± SEM (*P < 0.05, **P < 0.005, ***P < 0.001) of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862428&req=5

f0003: CaMKII acts as a downstream component of Hh-Cad99C, which required for DUOX activity. (A) Uracil-induced DUOX activation was abolished in CaMKII-KD flies. Uracil (20 nM) was administered orally to 3-day-old adult flies for 90 min, and DUOX-dependent ROS production in the midgut was visualized by HOCl−specific R19S dye (orange). Representative confocal microscopic images and the percentage of ROS-positive intestines is shown. (B) Epistatic analyses. Constitutive ROS generation found in flies carrying constitutive Hh signaling (Cos2-KD) can be abolished in the presence of CaMKII-KD. (C) CaMKII is required for uracil-induced Ca2+ mobilization. FRET analysis for the measurement of Ca2+ using flies expressing cameleon calcium sensor was performed. FRET efficiency (E %) was expressed as the mean ± SD from at least 30 flies. (D) Forced calcium increase is sufficient to induce DUOX activation in a CaMKII-KD animals. Flies over-expressing dominant-negative form of sarco/endoplasmic reticulum Ca2+-ATPase (SercaDN) were used. Data in A-D were analyzed using an ANOVA followed by Tukey post hoc test (C) or by Tamhane's T2 post hoc test (A,B and D); values represent mean ± SEM (*P < 0.05, **P < 0.005, ***P < 0.001) of at least three independent experiments.
Mentions: To determine whether the high infection-induced lethality observed in CaMKII-KD flies was due to impaired DUOX-dependent ROS generation, we measured uracil-induced ROS generation in these animals. We found that CaMKII-KD animals failed to generate ROS following uracil ingestion (Fig. 3A), indicating that CaMKII is required for DUOX activation. Epistatic analyses further revealed that constitutive ROS generation found in flies carrying constitutive Hh signaling could be abolished in the presence of CaMKII-KD (Fig. 3B). These results indicate that CaMKII acts as a downstream component of Hh signaling.Figure 3.

Bottom Line: We have recently found that uracil stimulates Hedgehog signaling, which in turn upregulates cadherin99C (Cad99C) expression in enterocytes.These observations illustrate the complexity of signaling cascades in uracil-induced signaling pathways.Moreover, we will provide a brief discussion on two different models for uracil recognition and uracil-induced DUOX activation in Drosophila enterocytes.

View Article: PubMed Central - PubMed

Affiliation: a School of Biological Science, Seoul National University and National Creative Research Initiative Center for Hologenomics, Seoul National University , Seoul , South Korea.

ABSTRACT
Intestinal dual oxidase (DUOX) activation is the first line of host defense against enteric infection in Drosophila. DUOX enzymatic activity is mainly controlled by phospholipase C-β (PLCβ)-dependent calcium mobilization, whereas DUOX gene expression is mainly controlled by the MEKK1-p38 mitogen-activated protein kinase pathway. Furthermore, bacterial-derived uracil molecules act as ligands for DUOX activation. However, our current understanding of uracil-induced signal transduction pathways remain incomplete. We have recently found that uracil stimulates Hedgehog signaling, which in turn upregulates cadherin99C (Cad99C) expression in enterocytes. Cad99C molecules, along with PLCβ and protein kinase C, induce the formation of signaling endosomes that facilitate intracellular calcium mobilization for DUOX activity. These observations illustrate the complexity of signaling cascades in uracil-induced signaling pathways. Here, we further demonstrated the role of lipid raft formation and calmodulin-dependent protein kinase-II on endosome formation and calcium mobilization, respectively. Moreover, we will provide a brief discussion on two different models for uracil recognition and uracil-induced DUOX activation in Drosophila enterocytes.

No MeSH data available.


Related in: MedlinePlus