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A non-canonical cGAS–STING–PERK pathway facilitates the translational program critical for senescence and organ fibrosis

Abstract

Innate DNA sensing via the cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS–STING) mechanism surveys microbial invasion and cellular damage and thus participates in various human infectious diseases, autoimmune diseases and cancers. However, how DNA sensing rapidly and adaptively shapes cellular physiology is incompletely known. Here we identify the STING–PKR-like endoplasmic reticulum kinase (PERK)–eIF2α pathway, a previously unknown cGAS–STING mechanism, enabling an innate immunity control of cap-dependent messenger RNA translation. Upon cGAMP binding, STING at the ER binds and directly activates the ER-located kinase PERK via their intracellular domains, which precedes TBK1–IRF3 activation and is irrelevant to the unfolded protein response. The activated PERK phosphorylates eIF2α, forming an inflammatory- and survival-preferred translation program. Notably, this STING–PERK–eIF2α pathway is evolutionarily primitive and physiologically critical to cellular senescence and organ fibrosis. Pharmacologically or genetically targeting this non-canonical cGAS–STING pathway attenuated lung and kidney fibrosis. Collectively, the findings identify an alternative innate immune pathway and its critical role in organ fibrosis, report an innate immunity-directed translation program and suggest the therapeutic potential for targeting the STING–PERK pathway in treating fibrotic diseases.

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Fig. 1: STING inhibits cap-dependent translation via the PERK–eIF2α axis, independent of the UPR.
Fig. 2: Activation of STING–PERK–eIF2α signalling precedes that of STING–TBK1–IRF3.
Fig. 3: STING interacts with and directly activates PERK via its intracellular CTD.
Fig. 4: PERK–eIF2α signalling differs from cascades of TBK1–IRF3, NF-κB signalling and autophagy.
Fig. 5: STING facilitates translation programs for inflammatory and survival signalling.
Fig. 6: The STING–PERK–eIF2α axis is critical for damage-induced senescence.
Fig. 7: Intervening STING–PERK–eIF2α signalling ameliorates the progression of fibrotic pulmonary diseases.
Fig. 8: The STING–PERK–eIF2α cascade drives pulmonary and renal fibrosis.

Data availability

Polysome-seq data have been deposited in the NCBI Gene Expression Omnibus database under accession code GSE165910. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We are grateful to J. Han, J. Shao and Z. Jiang for the reagents and Y. J. Zhang for helpful discussions. This research was sponsored by the NSFC Projects (31830052 and 31725017 to P.X.) and the National Key Research and Development Program of China (2021YFA1301401 to P.X.). Thanks also go to the Life Sciences Institute core facilities of Zhejiang University for technical assistance.

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D.Z. and Y.L. carried out most of the experiments. Y.Z., Q.Z., H.G., S.L., S.C., C.M., C.C., Z.L., Y.X., S.O., X.-H.F., T.L. and L.S. contributed to several experiments and helped with data analyses and discussions. P.X. and D.Z. conceived the study and designed the experiments. P.X. wrote the manuscript.

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Correspondence to Tingbo Liang, Li Shen or Pinglong Xu.

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Extended data

Extended Data Fig. 1 STING inhibits cap-dependent translation through the PERK-eIF2α axis and is independent of the UPR.

(A) Expression of STING but not MAVS inhibited new protein synthesis, as determined by immunoblotting for puromycin in HEK293 cells. The graph shows the relative protein synthesis rate normalized to that of GAPDH. n = 3 independent experiments (mean ± SEM), and P values were indicated by one-way ANOVA with the Bonferroni correction. (B) Global protein synthesis was substantially attenuated by inducing expression of a constitutively active form of STING (R281Q) in DLD1 cells via the Tet-On system, as indicated by the declined levels of puromycin. (C) Translation arrest induced by the ER and oxidative stress was typical in STING-deficient HCT116 cells. (D) Ectopic expression of both NvSTING and DrSTING distinctly induced eIF2α but not IRF3 phosphorylation in HEK293 cells. (E) Phosphorylation of endogenous eIF2α in HEK293 cells was induced by ectopic expression of hSTING, NvSTING, or DmSTING. (F-G) Immunofluorescence imaging revealed that eIF2α was mainly localized in the cytoplasm and partially associated with the ER, which was not aggregated upon STING expression. Scale bars=20 μm. (H) eIF2α phosphorylation, which STING triggered, was declined explicitly in the presence of a PERK inhibitor but not an ER stress inhibitor (TUDCA), TBK1 inhibitor (BX795), or mTOR inhibitor (rapamycin) in HCT116 cells. (I) Expression of STING led to eIF2α phosphorylation in HCT116 cells, which was abolished by PERK inhibitors (iPERK-1 and iPERK-2) but not NF-κB inhibitors (TPCA-1 and JSH-23). (J) Ectopic expression of STING R331A/R334A failed to induce the phosphorylation of both eIF2α and IRF3 in HEK293 cells. (K) Immunofluorescence imaging revealed the partial accumulation of ATF6, an indication of ATF6 activation, by the treatment of UPR activator but nut STING agonist. Scale bars=10 μm. (L) Genetic ablation of STING expression did not affect the activation of UPR signalling, as evidenced by the similar levels of phospho-PERK, phospho-eIF2α, and phospho-IRE1α in WT and STING KO MEFs upon DTT treatment. Data shown in A-L represent three independent experiments.

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Extended Data Fig. 2 Activation of STING-PERK-eIF2α signalling precedes that of the STING-TBK1-IRF3 cascade.

(A) In the presence of TBK1 inhibitor, ectopic expression of STING in HEK293 cells failed to induce IRF3 phosphorylation, but its capability to induce eIF2α phosphorylation remained intact. (B) STING activation in primary MEFs, as induced by agonist diABZI, led to PERK-eIF2α signalling activation, as indicated by immunoblotting for phospho-PERK. Data in a and b represent three independent experiments.

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Extended Data Fig. 3 STING interacts with and directly activates PERK via its intracellular C-terminal domain.

(A) Coimmunoprecipitation assays showed that the interacting motifs, that is, the C-terminal region of the PERK kinase domain, were sufficient for STING interaction. (B) Immunofluorescence imaging revealed that PERK ICD and its kinase-dead mutant were mainly localized in the cytoplasm and probably associated with cytoskeleton and plasma membrane. Scale bars=10 μm. (C) PERK KO HEK293 cells were generated via a CRISPR-based strategy and verified by immunoblotting for PERK. Data in A-C represent three independent experiments.

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Extended Data Fig. 4 The PERK-eIF2α cascade is discrete with TBK1-IRF3 and NF-κB signalling and STING-induced autophagy.

(A) Ectopic expression of STING CTD induced the phosphorylation of endogenous eIF2α in HEK293 cells in a dose-dependent manner. (B) Immunofluorescence imaging revealed that STING CTD was distributed in the cytoplasm and the nucleus, distinct from the full-length STING that associates the ER. Scale bars=20 μm. (C) Treatment of PERK inhibitor unaffected the STING-induced autophagy in DLD1 cells, as indicated by LC3 lipidation. Data in A-C represent three independent experiments. (D-E) PERK inhibition did not substantially regulate STING-induced NF-κB signalling activation, either STING versions from human (D), anemone (E), or Drosophila (E), as evidenced by the reporter assays. In D-E, n = 3 independent experiments (mean ± SEM), and P values were indicated by one-way ANOVA with the Bonferroni correction.

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Extended Data Fig. 5 STING-initiated translation program selectively facilitates inflammatory and survival signalling.

(A) The mRNA levels of DDX21 and CDC25B were not notably altered upon the treatment of cGAMP at 1 h, when their protein levels were enhanced. n = 3 independent experiments (mean ± SEM), and P values were indicated by one-way ANOVA with the Bonferroni correction.

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Extended Data Fig. 6 The STING-PERK-eIF2α axis is critical for damage-induced cellular senescence.

(A) HU-induced senescence was substantially attenuated upon the treatments of PERK inhibitor but not inhibitors of IKKβ or the UPR, as revealed by SA-β-Gal staining. (B-C) PERK ablation in HCT116 cells via a CRISPR-based strategy significantly suppressed HU-induced senescence, as evidenced by SA-β-Gal staining (A). This suppression was reversed upon reconstitution of murine PERK (B). The KO and reconstitution efficiencies were verified by immunoblotting (bottom panel, B), and statistics were calculated for SA-β-Gal-stained cells (right panel, A). (D) The PERK-deficient cell population was generated by CRISPR-mediated genome editing, and its efficiency was determined by immunoblotting. (E) Perk ablation in primary MEFs led to a decreased expression of p21Waf1, a key indicator of cellular senescence. (F) Reconstitution of STING-deficient HCT116 cells with the 2LA mutant did not restore HU-induced apoptosis, as revealed by endogenous cleaved PARP proteins. Scale bar=100 μm (A, B, C). In A, B, and E, n = 3 independent experiments (mean ± SEM), and P values were indicated by one-way ANOVA with the Bonferroni correction. Data in C, D, and F represent three independent experiments.

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Extended Data Fig. 7 Intervening the STING-PERK-eIF2α axis ameliorates the progression of fibrotic pulmonary diseases.

(A) Pulmonary fibrosis was induced by bleomycin in WT and STING KO C57BL/6 mice (top panel). Genetic ablation of STING substantially ameliorated lung fibrosis symptoms, as evidenced by the diminished areas of collagen and declined Ashcroft fibrosis scores. Scale bars=100 μm. Data shown represent mean ± SEM (n = 4 mice in the saline group; n = 8 mice in the other groups), and P values were indicated by one-way ANOVA with the Bonferroni correction. (B-C) Bleomycin-induced pulmonary fibrosis was markedly attenuated in STING-deficient mice (B) or upon the treatment of PERK inhibitor (C), as revealed by immunoblotting for the reduced levels of α-SMA and collagen I. (D) Schematic figure showed the design, generation, and verification of the Eif2ak3 heterozygotes, and the gRNA target sequence (5’-TGGACAGGAGGTGCCTCGTTGGG-3’) was located in the exon 1 of murine Eif2ak3 gene. (E) PERK heterozygotes (Eif2ak3 + /-) were largely normal in appearance and weight and were fertile, while its homozygotes were embryonic lethal. (F) Bleomycin-induced pulmonary fibrosis was markedly attenuated in PERK half-deficiency mice, as revealed by immunoblotting for α-SMA and collagen I. (G) Immunoblottings for α-SMA and collagen I showed that Bleomycin-induced pulmonary fibrosis was severely prevented by PERK inhibition, but not the inhibition of the UPR. Images of lung sections for individual mice in Extended Data Fig. 7 were provided in Supplementary Information. Data in B, C, F and G represent three independent experiments.

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Extended Data Fig. 8 The STING-PERK-eIF2α cascade underlies pulmonary and renal fibrosis.

(A-D) Immunofluorescence imaging showed the cGAMP-triggered upregulation of endogenous α-SMA and collagen I proteins in lung sections, which were substantially dampened in PERK half-deficiency mice (A-B), or upon the treatment of the PERK inhibitor iPERK-2 (C-D). Scale bars=100 μm. Data in A-D represent three independent experiments. (E) As expected, the kidney collected from the UUO surgery group on day 14 was dilated. Scale bars=1 cm. Data shown represent n = 13 mice in the sham group; n = 16 mice in other groups. (F) A schematic figure describing the signalling mechanism and function of this non-canonical cGAS-STING-PERK pathway, which occurred at the ER to regulate mRNA translation and preceded to the classical STING-TBK1-IRF3 cascade that regulates mRNA transcription.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Reporting Summary

Supplementary Tables

Supplementary Table 1: List of recombinant DNA. Supplementary Table 2: Antibodies used in the study. Supplementary Table 3: Oligos used in the study. Supplementary Table 4: Quality control of polysome-seq samples

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Zhang, D., Liu, Y., Zhu, Y. et al. A non-canonical cGAS–STING–PERK pathway facilitates the translational program critical for senescence and organ fibrosis. Nat Cell Biol 24, 766–782 (2022). https://doi.org/10.1038/s41556-022-00894-z

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