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Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

Lee JE, Oney M, Frizzell K, Phadnis N, Hollien J - G3 (Bethesda) (2015)

Bottom Line: The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh).Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress.Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, Utah 84112.

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Atf4 binding sites within the promoters of Ldh and Pfk mediate Atf4-dependent transcriptional up-regulation. (A) We stably transfected S2 cells with the GFP reporter constructs diagrammed on the left. We then mock-treated or depleted cells of Atf4 by RNAi, incubated with and without DTT (2 mM, 5 hr), and measured relative GFP RNA levels by qPCR. (B) We transiently transfected S2 cells with reporter constructs containing wild-type or mutated promoter sequences as shown on the left. We then incubated cells with or without DTT (2 mM, 5 hr) and measured relative GFP RNA levels by qPCR. For all panels: shown are the means ± SDs of at least three independent experiments. *P < 0.05; **P < 0.005; ***P < 0.001, Student’s paired t-test. Atf4, activating transcription factor 4; dsRNA, double-stranded RNA; DTT, dithiothreitol; GFP, green fluorescent protein; Ldh, Lactate dehydrogenase; Pfk, phosphofructokinase; qPCR, quantitative polymerase chain reaction; RNAi, RNA interference; TCA, tricarboxylic acid; Tm, tunicamycin
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fig5: Atf4 binding sites within the promoters of Ldh and Pfk mediate Atf4-dependent transcriptional up-regulation. (A) We stably transfected S2 cells with the GFP reporter constructs diagrammed on the left. We then mock-treated or depleted cells of Atf4 by RNAi, incubated with and without DTT (2 mM, 5 hr), and measured relative GFP RNA levels by qPCR. (B) We transiently transfected S2 cells with reporter constructs containing wild-type or mutated promoter sequences as shown on the left. We then incubated cells with or without DTT (2 mM, 5 hr) and measured relative GFP RNA levels by qPCR. For all panels: shown are the means ± SDs of at least three independent experiments. *P < 0.05; **P < 0.005; ***P < 0.001, Student’s paired t-test. Atf4, activating transcription factor 4; dsRNA, double-stranded RNA; DTT, dithiothreitol; GFP, green fluorescent protein; Ldh, Lactate dehydrogenase; Pfk, phosphofructokinase; qPCR, quantitative polymerase chain reaction; RNAi, RNA interference; TCA, tricarboxylic acid; Tm, tunicamycin

Mentions: We transfected S2 cells with 2 μg of plasmid using Cellfectin II (Invitrogen). For polyclonal stable cell lines (as in Figure 3D and Figure 5A), we cotransfected our expression plasmids (1.8 μg) with a hygromycin or puromycin resistance plasmid (0.2 μg) and selected for resistant cells. For Atf4 overexpression studies (Figure 3D), we induced expression with CuSO4 (250 μM, 36 hr) before collecting RNA samples. For promoter constructs, we treated control cells transfected with pMT-GFP with CuSO4 (250 μM, 16 hr) before inducing ER stress with DTT as described above. Finally, for transiently transfected cells (Figure 5B), we allowed cells to recover overnight before inducing ER stress.


Drosophila melanogaster activating transcription factor 4 regulates glycolysis during endoplasmic reticulum stress.

Lee JE, Oney M, Frizzell K, Phadnis N, Hollien J - G3 (Bethesda) (2015)

Atf4 binding sites within the promoters of Ldh and Pfk mediate Atf4-dependent transcriptional up-regulation. (A) We stably transfected S2 cells with the GFP reporter constructs diagrammed on the left. We then mock-treated or depleted cells of Atf4 by RNAi, incubated with and without DTT (2 mM, 5 hr), and measured relative GFP RNA levels by qPCR. (B) We transiently transfected S2 cells with reporter constructs containing wild-type or mutated promoter sequences as shown on the left. We then incubated cells with or without DTT (2 mM, 5 hr) and measured relative GFP RNA levels by qPCR. For all panels: shown are the means ± SDs of at least three independent experiments. *P < 0.05; **P < 0.005; ***P < 0.001, Student’s paired t-test. Atf4, activating transcription factor 4; dsRNA, double-stranded RNA; DTT, dithiothreitol; GFP, green fluorescent protein; Ldh, Lactate dehydrogenase; Pfk, phosphofructokinase; qPCR, quantitative polymerase chain reaction; RNAi, RNA interference; TCA, tricarboxylic acid; Tm, tunicamycin
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390581&req=5

fig5: Atf4 binding sites within the promoters of Ldh and Pfk mediate Atf4-dependent transcriptional up-regulation. (A) We stably transfected S2 cells with the GFP reporter constructs diagrammed on the left. We then mock-treated or depleted cells of Atf4 by RNAi, incubated with and without DTT (2 mM, 5 hr), and measured relative GFP RNA levels by qPCR. (B) We transiently transfected S2 cells with reporter constructs containing wild-type or mutated promoter sequences as shown on the left. We then incubated cells with or without DTT (2 mM, 5 hr) and measured relative GFP RNA levels by qPCR. For all panels: shown are the means ± SDs of at least three independent experiments. *P < 0.05; **P < 0.005; ***P < 0.001, Student’s paired t-test. Atf4, activating transcription factor 4; dsRNA, double-stranded RNA; DTT, dithiothreitol; GFP, green fluorescent protein; Ldh, Lactate dehydrogenase; Pfk, phosphofructokinase; qPCR, quantitative polymerase chain reaction; RNAi, RNA interference; TCA, tricarboxylic acid; Tm, tunicamycin
Mentions: We transfected S2 cells with 2 μg of plasmid using Cellfectin II (Invitrogen). For polyclonal stable cell lines (as in Figure 3D and Figure 5A), we cotransfected our expression plasmids (1.8 μg) with a hygromycin or puromycin resistance plasmid (0.2 μg) and selected for resistant cells. For Atf4 overexpression studies (Figure 3D), we induced expression with CuSO4 (250 μM, 36 hr) before collecting RNA samples. For promoter constructs, we treated control cells transfected with pMT-GFP with CuSO4 (250 μM, 16 hr) before inducing ER stress with DTT as described above. Finally, for transiently transfected cells (Figure 5B), we allowed cells to recover overnight before inducing ER stress.

Bottom Line: The unfolded protein response transcription factor Atf4 was necessary for the up-regulation of glycolytic enzymes and Lactate dehydrogenase (Ldh).Finally, flies up-regulated Ldh and produced more lactate when subjected to ER stress.Together, these results suggest that Atf4 mediates a shift from a metabolism based on oxidative phosphorylation to one more heavily reliant on glycolysis, reminiscent of aerobic glycolysis or the Warburg effect observed in cancer and other proliferative cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, Utah 84112.

Show MeSH
Related in: MedlinePlus