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Recognition of cytosolic DNA attenuates glucose metabolism and induces AMPK mediated energy stress response.

Zheng M, Xie L, Liang Y, Wu S, Xu H, Zhang Y, Liu H, Lin D, Han J, Lu K - Int. J. Biol. Sci. (2015)

Bottom Line: Recognition of cytosolic DNA activates a series of cellular responses, including induction of pro-inflammatory genes such as type I interferon through the well-known cGAS-STING pathway.Here we show for the first time that intracellular administration of either single or double stranded interferon stimulating DNA (ISD), but not poly(dA) suppresses cell growth in many different cell types.Suppression of cell growth by cytosolic DNA is cGAS/STING independent and associated with inhibition of glucose metabolism, ATP depletion and subsequent cellular energy stress responses including activation of AMPK and inactivation of mTORC1.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, 350108, China. ; 2. Translational Medicine Institute, Fujian Medical University, Fuzhou, Fujian, 350108, China.

ABSTRACT
Both viral infection and DNA transfection expose single-stranded or double-stranded DNA to the cytoplasm of mammalian cells. Recognition of cytosolic DNA activates a series of cellular responses, including induction of pro-inflammatory genes such as type I interferon through the well-known cGAS-STING pathway. Here we show for the first time that intracellular administration of either single or double stranded interferon stimulating DNA (ISD), but not poly(dA) suppresses cell growth in many different cell types. Suppression of cell growth by cytosolic DNA is cGAS/STING independent and associated with inhibition of glucose metabolism, ATP depletion and subsequent cellular energy stress responses including activation of AMPK and inactivation of mTORC1. Our results suggest that in concert with but independent of innate immune response, recognition of cytosolic DNA induced cellular energy stress potentially functions as a metabolic barrier to viral replication.

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Both ssISD and dsISD induce acute metabolic stress in nucleic acid type, sequence and does dependent manner. (A) 293T cells were transfected with ssISD, dsISD, poly(dA) or poly(I:C). WST-1 assay was performed at indicated time points. (B) 293T cells were transfected as in (A) with indicated does. 6 hours post-transfection, WST-1 assay was performed. (C) 293T cells were transfected with dsISD, ssISD or poly(dA). CCK-8 assay was performed at 6 hours post-transfection. (D) 293T cells were transfected as indicated and WST-1 assay was taken 6 hours post transfection. ss: single stranded. ds: double stranded. Data of three independent replicates are presented as the mean +/- s.e.m., n=3.
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Figure 2: Both ssISD and dsISD induce acute metabolic stress in nucleic acid type, sequence and does dependent manner. (A) 293T cells were transfected with ssISD, dsISD, poly(dA) or poly(I:C). WST-1 assay was performed at indicated time points. (B) 293T cells were transfected as in (A) with indicated does. 6 hours post-transfection, WST-1 assay was performed. (C) 293T cells were transfected with dsISD, ssISD or poly(dA). CCK-8 assay was performed at 6 hours post-transfection. (D) 293T cells were transfected as indicated and WST-1 assay was taken 6 hours post transfection. ss: single stranded. ds: double stranded. Data of three independent replicates are presented as the mean +/- s.e.m., n=3.

Mentions: In order to better characterize ISD-induced cellular response, we performed time-course and dose-response analyses of ISD using WST-1 assay. Interestingly, as early as 4 hours after transfection, ssISD or dsISD dramatically decreased WST-1 reduction in a dose-dependent manner (Fig. 2A and 2B). No significant difference was observed between the sense strand and anti-sense strand of ssISD (Supplementary Figure 2). In contrast, poly(dA) or poly(I:C) (a dsRNA mimic) had little effect under the same conditions (Fig. 2A and 2B). Similar results were obtained using either conventional MTT assay (Supplementary Figure 3) or newly developed CCK-8 assay, which is more stable and sensitive (Fig. 2C). Next, we tested synthetic DNAs comprising different kinds of sequences, including poly(dT), poly(dA-dT) and poly(dG-dC). Both single stranded and double stranded poly(dA-dT) and poly(dG-dC), but not poly(dT) had the effect (Fig. 2D). We also tested the cell type specificity of this phenotype. In addition to immortalized human 293 cells, ISD-induced cellular response was also observed in many other cell lines including human cancerous THP-1 cells and immortalized mouse embryo fibroblast (MEF) cells (Supplementary Figure. 4A and 4B). Combined with PMS (phenazine methosulfate) electron mediator, WST-1 is readily reduced by NAD(P)H-dependent oxidoreductases in the cell into water-soluble formazan dyes 18-20. Therefore, WST-1 reduction is proportional to intracellular reducing power like NAD(P)H level, reflecting the cellular metabolic viability. Thus, these data suggest that both ssISD and dsISD induce acute metabolic stress in nucleic acids type, sequence and dose dependent manner.


Recognition of cytosolic DNA attenuates glucose metabolism and induces AMPK mediated energy stress response.

Zheng M, Xie L, Liang Y, Wu S, Xu H, Zhang Y, Liu H, Lin D, Han J, Lu K - Int. J. Biol. Sci. (2015)

Both ssISD and dsISD induce acute metabolic stress in nucleic acid type, sequence and does dependent manner. (A) 293T cells were transfected with ssISD, dsISD, poly(dA) or poly(I:C). WST-1 assay was performed at indicated time points. (B) 293T cells were transfected as in (A) with indicated does. 6 hours post-transfection, WST-1 assay was performed. (C) 293T cells were transfected with dsISD, ssISD or poly(dA). CCK-8 assay was performed at 6 hours post-transfection. (D) 293T cells were transfected as indicated and WST-1 assay was taken 6 hours post transfection. ss: single stranded. ds: double stranded. Data of three independent replicates are presented as the mean +/- s.e.m., n=3.
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Related In: Results  -  Collection

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Figure 2: Both ssISD and dsISD induce acute metabolic stress in nucleic acid type, sequence and does dependent manner. (A) 293T cells were transfected with ssISD, dsISD, poly(dA) or poly(I:C). WST-1 assay was performed at indicated time points. (B) 293T cells were transfected as in (A) with indicated does. 6 hours post-transfection, WST-1 assay was performed. (C) 293T cells were transfected with dsISD, ssISD or poly(dA). CCK-8 assay was performed at 6 hours post-transfection. (D) 293T cells were transfected as indicated and WST-1 assay was taken 6 hours post transfection. ss: single stranded. ds: double stranded. Data of three independent replicates are presented as the mean +/- s.e.m., n=3.
Mentions: In order to better characterize ISD-induced cellular response, we performed time-course and dose-response analyses of ISD using WST-1 assay. Interestingly, as early as 4 hours after transfection, ssISD or dsISD dramatically decreased WST-1 reduction in a dose-dependent manner (Fig. 2A and 2B). No significant difference was observed between the sense strand and anti-sense strand of ssISD (Supplementary Figure 2). In contrast, poly(dA) or poly(I:C) (a dsRNA mimic) had little effect under the same conditions (Fig. 2A and 2B). Similar results were obtained using either conventional MTT assay (Supplementary Figure 3) or newly developed CCK-8 assay, which is more stable and sensitive (Fig. 2C). Next, we tested synthetic DNAs comprising different kinds of sequences, including poly(dT), poly(dA-dT) and poly(dG-dC). Both single stranded and double stranded poly(dA-dT) and poly(dG-dC), but not poly(dT) had the effect (Fig. 2D). We also tested the cell type specificity of this phenotype. In addition to immortalized human 293 cells, ISD-induced cellular response was also observed in many other cell lines including human cancerous THP-1 cells and immortalized mouse embryo fibroblast (MEF) cells (Supplementary Figure. 4A and 4B). Combined with PMS (phenazine methosulfate) electron mediator, WST-1 is readily reduced by NAD(P)H-dependent oxidoreductases in the cell into water-soluble formazan dyes 18-20. Therefore, WST-1 reduction is proportional to intracellular reducing power like NAD(P)H level, reflecting the cellular metabolic viability. Thus, these data suggest that both ssISD and dsISD induce acute metabolic stress in nucleic acids type, sequence and dose dependent manner.

Bottom Line: Recognition of cytosolic DNA activates a series of cellular responses, including induction of pro-inflammatory genes such as type I interferon through the well-known cGAS-STING pathway.Here we show for the first time that intracellular administration of either single or double stranded interferon stimulating DNA (ISD), but not poly(dA) suppresses cell growth in many different cell types.Suppression of cell growth by cytosolic DNA is cGAS/STING independent and associated with inhibition of glucose metabolism, ATP depletion and subsequent cellular energy stress responses including activation of AMPK and inactivation of mTORC1.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian, 350108, China. ; 2. Translational Medicine Institute, Fujian Medical University, Fuzhou, Fujian, 350108, China.

ABSTRACT
Both viral infection and DNA transfection expose single-stranded or double-stranded DNA to the cytoplasm of mammalian cells. Recognition of cytosolic DNA activates a series of cellular responses, including induction of pro-inflammatory genes such as type I interferon through the well-known cGAS-STING pathway. Here we show for the first time that intracellular administration of either single or double stranded interferon stimulating DNA (ISD), but not poly(dA) suppresses cell growth in many different cell types. Suppression of cell growth by cytosolic DNA is cGAS/STING independent and associated with inhibition of glucose metabolism, ATP depletion and subsequent cellular energy stress responses including activation of AMPK and inactivation of mTORC1. Our results suggest that in concert with but independent of innate immune response, recognition of cytosolic DNA induced cellular energy stress potentially functions as a metabolic barrier to viral replication.

Show MeSH
Related in: MedlinePlus