Limits...
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.

Show MeSH

Related in: MedlinePlus

Cytosolic ISD induces ATP depletion and AMPK mediated energy stress response. (A) Intracellular ATP level was measured at indicated time points. (B, C) 293T cells were transfected as indicated. 6 hours post-transfection, ATP level was measured (B) and total cell lysate was blotted with anti phosphor-AMPKα(T172), AMPKα, phosphor-AMPKβ(S108), AMPKβ, phosphor-Ratpor (S792), phosphor-S6K1(T389), LC3, PKM2 and GAPDH antibodies, respectively(C). F: sense strand. R: anti-sense strand. Data of three independent replicates are presented as the mean +/- s.e.m., n=3.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4400389&req=5

Figure 6: Cytosolic ISD induces ATP depletion and AMPK mediated energy stress response. (A) Intracellular ATP level was measured at indicated time points. (B, C) 293T cells were transfected as indicated. 6 hours post-transfection, ATP level was measured (B) and total cell lysate was blotted with anti phosphor-AMPKα(T172), AMPKα, phosphor-AMPKβ(S108), AMPKβ, phosphor-Ratpor (S792), phosphor-S6K1(T389), LC3, PKM2 and GAPDH antibodies, respectively(C). F: sense strand. R: anti-sense strand. Data of three independent replicates are presented as the mean +/- s.e.m., n=3.

Mentions: Glucose metabolism is a major source of intracellular energy supply. We thus asked whether recognition of cytosolic DNA also lead to ATP depletion. Indeed, ssISD significantly reduced intracellular ATP levels in a time-dependent manner starting at 4 hours after transfection (Fig. 6A) and this phenotype was again induced only by dsISD or ssISD, but not poly(dA) (Fig. 6B). To examine whether ISD-induced ATP depletion activates cellular energy stress response pathways biochemically, we assayed the AMPK/mTORC1/S6K1 pathway because AMPK is a well known energy stress sensor in the cell23 and its activation leads to inhibition of mTORC1 by phosphorylation of Raptor on S792, leading to reduced S6K1 phosphorylation on T38924. As expected, both ssISD and dsISD, but not poly(dA) induced AMPKα phosphorylation on T172 and AMPKβ phosphorylation on S108, accompanied with increased Raptor phosphorylation on S792 and reduced S6K1 phosphorylation on T389 (Fig. 6C). These results support the notion that inhibition of glucose metabolism induced by cytosolic DNA also leads to ATP depletion and AMPK mediated cellular energy stress response.


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)

Cytosolic ISD induces ATP depletion and AMPK mediated energy stress response. (A) Intracellular ATP level was measured at indicated time points. (B, C) 293T cells were transfected as indicated. 6 hours post-transfection, ATP level was measured (B) and total cell lysate was blotted with anti phosphor-AMPKα(T172), AMPKα, phosphor-AMPKβ(S108), AMPKβ, phosphor-Ratpor (S792), phosphor-S6K1(T389), LC3, PKM2 and GAPDH antibodies, respectively(C). F: sense strand. R: anti-sense strand. Data of three independent replicates are presented as the mean +/- s.e.m., n=3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4400389&req=5

Figure 6: Cytosolic ISD induces ATP depletion and AMPK mediated energy stress response. (A) Intracellular ATP level was measured at indicated time points. (B, C) 293T cells were transfected as indicated. 6 hours post-transfection, ATP level was measured (B) and total cell lysate was blotted with anti phosphor-AMPKα(T172), AMPKα, phosphor-AMPKβ(S108), AMPKβ, phosphor-Ratpor (S792), phosphor-S6K1(T389), LC3, PKM2 and GAPDH antibodies, respectively(C). F: sense strand. R: anti-sense strand. Data of three independent replicates are presented as the mean +/- s.e.m., n=3.
Mentions: Glucose metabolism is a major source of intracellular energy supply. We thus asked whether recognition of cytosolic DNA also lead to ATP depletion. Indeed, ssISD significantly reduced intracellular ATP levels in a time-dependent manner starting at 4 hours after transfection (Fig. 6A) and this phenotype was again induced only by dsISD or ssISD, but not poly(dA) (Fig. 6B). To examine whether ISD-induced ATP depletion activates cellular energy stress response pathways biochemically, we assayed the AMPK/mTORC1/S6K1 pathway because AMPK is a well known energy stress sensor in the cell23 and its activation leads to inhibition of mTORC1 by phosphorylation of Raptor on S792, leading to reduced S6K1 phosphorylation on T38924. As expected, both ssISD and dsISD, but not poly(dA) induced AMPKα phosphorylation on T172 and AMPKβ phosphorylation on S108, accompanied with increased Raptor phosphorylation on S792 and reduced S6K1 phosphorylation on T389 (Fig. 6C). These results support the notion that inhibition of glucose metabolism induced by cytosolic DNA also leads to ATP depletion and AMPK mediated cellular energy stress response.

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