Attenuation of c GAS ‐ STING signaling is mediated by a p62/ SQSTM 1‐dependent autophagy pathway activated by TBK1
2018; Springer Nature; Volume: 37; Issue: 8 Linguagem: Inglês
10.15252/embj.201797858
ISSN1460-2075
AutoresThaneas Prabakaran, Chiranjeevi Bodda, Christian Krapp, Bao‐cun Zhang, Maria H Christensen, Chenglong Sun, Line S. Reinert, Yujia Cai, Søren B. Jensen, Morten Kelder Skouboe, Jens R. Nyengaard, Craig B. Thompson, Robert Jan Lebbink, Ganes C. Sen, Geert Loo, Rikke Nielsen, Masaaki Komatsu, Lene N. Nejsum, Martin R. Jakobsen, Mads Gyrd‐Hansen, Søren R. Paludan,
Tópico(s)Cancer-related molecular mechanisms research
ResumoArticle1 March 2018free access Transparent process Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1 Thaneas Prabakaran Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Chiranjeevi Bodda Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Nuffield Department of Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Christian Krapp Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Bao-cun Zhang Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Maria H Christensen Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Chenglong Sun Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Line Reinert Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Yujia Cai Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Søren B Jensen Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Morten K Skouboe Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Jens R Nyengaard Department of Clinical Medicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Craig B Thompson Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA Search for more papers by this author Robert Jan Lebbink Medical Microbiology, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Ganes C Sen Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Geert van Loo Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Rikke Nielsen Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Masaaki Komatsu Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Search for more papers by this author Lene N Nejsum Department of Clinical Medicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Martin R Jakobsen Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Mads Gyrd-Hansen orcid.org/0000-0001-5641-5019 Nuffield Department of Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Søren R Paludan Corresponding Author [email protected] orcid.org/0000-0001-9180-4060 Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Thaneas Prabakaran Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Chiranjeevi Bodda Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Nuffield Department of Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Christian Krapp Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Bao-cun Zhang Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Maria H Christensen Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Chenglong Sun Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Line Reinert Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Yujia Cai Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Søren B Jensen Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Morten K Skouboe Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Jens R Nyengaard Department of Clinical Medicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Craig B Thompson Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA Search for more papers by this author Robert Jan Lebbink Medical Microbiology, University Medical Center, Utrecht, The Netherlands Search for more papers by this author Ganes C Sen Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Search for more papers by this author Geert van Loo Inflammation Research Center, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Rikke Nielsen Department of Biomedicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Masaaki Komatsu Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Search for more papers by this author Lene N Nejsum Department of Clinical Medicine, Aarhus University, Aarhus, Denmark Search for more papers by this author Martin R Jakobsen Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Mads Gyrd-Hansen orcid.org/0000-0001-5641-5019 Nuffield Department of Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Søren R Paludan Corresponding Author [email protected] orcid.org/0000-0001-9180-4060 Department of Biomedicine, Aarhus University, Aarhus, Denmark Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Author Information Thaneas Prabakaran1,2, Chiranjeevi Bodda1,2,3, Christian Krapp1,2, Bao-cun Zhang1,2, Maria H Christensen1,2, Chenglong Sun1,2, Line Reinert1,2, Yujia Cai1,2, Søren B Jensen1,2, Morten K Skouboe1,2, Jens R Nyengaard4, Craig B Thompson5, Robert Jan Lebbink6, Ganes C Sen7, Geert Loo8,9, Rikke Nielsen1, Masaaki Komatsu10, Lene N Nejsum4, Martin R Jakobsen1,2, Mads Gyrd-Hansen3 and Søren R Paludan *,1,2 1Department of Biomedicine, Aarhus University, Aarhus, Denmark 2Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark 3Nuffield Department of Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK 4Department of Clinical Medicine, Aarhus University, Aarhus, Denmark 5Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA 6Medical Microbiology, University Medical Center, Utrecht, The Netherlands 7Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA 8Inflammation Research Center, VIB, Ghent, Belgium 9Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 10Department of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan *Corresponding author. Tel: +45 2899 2066; E-mail: [email protected] EMBO J (2018)37:e97858https://doi.org/10.15252/embj.201797858 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING. Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response. Synopsis Stimulation of the cGAS-STING pathway by cytosolic DNA leads to STING ubiquitination and degradation. The downstream cGAS-STING kinase TBK1 also phosphorylates the selective autophagy receptor p62, which in turn directs STING for degradation by autophagy. Abrogation of autophagy severely impaired DNA-stimulated STING degradation. p62 is essential for DNA-stimulated STING degradation. Cells lacking p62 have elevated interferon responses to cytoplasmic DNA and DNA pathogens. TBK1 phosphorylates p62, which promoted STING degradation and regulation of the pathway. Introduction Stimulus-dependent activation of immune responses is essential to achieve optimal tempo-spatial immune activities at sites of infection and to avoid tissue damage. To control immune reactions, cells harbor regulatory mechanisms that down-modulate immune-stimulatory signals (Chiao et al, 1994; Starr et al, 1997; Liew et al, 2005; Patel & Garcia-Sastre, 2014; Paludan, 2015). These mechanisms are important to avoid excess inflammation as evidenced by auto-inflammatory diseases in patient with loss-of-function mutations in genes encoding immune-regulatory proteins (Crow & Manel, 2015). Cytosolic DNA is highly immune-stimulatory and is detected by a panel of sensors to stimulate production of type I interferon (IFN)s, interleukin 1β, and activation of death pathways (Hornung et al, 2009; Unterholzner et al, 2010; Sun et al, 2013; Monroe et al, 2014). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is the key cytosolic DNA sensor that stimulates the IFN induction pathway (Li et al, 2013; Sun et al, 2013). Upon DNA binding, cGAS synthesizes 2′3′ cGAMP, which binds the adaptor protein stimulator of IFN genes (STING) and induces a conformational change in the STING dimer, thus enabling recruitment and activation of the tank-binding kinase (TBK)1 and the transcription factor IFN regulatory factor (IRF)3 (Paludan & Bowie, 2013). The induction of type I IFNs by DNA is essential for defense against virus infections and for anti-cancer immunity (Ishikawa et al, 2009; Li et al, 2013; Schoggins et al, 2013; Deng et al, 2014; Woo et al, 2014; Reinert et al, 2016). However, pathological roles have also been ascribed to type I IFNs in bacterial infections and in chronic viral infections (Auerbuch et al, 2004; Teijaro et al, 2013). Most notably, excess activation of the cGAS-STING pathway is associated with auto-inflammatory diseases, including Aicardi–Goutiéres Syndrome and systemic lupus erythematosus (Crow & Manel, 2015; Gall et al, 2012; Gao et al, 2015). To keep the cytoplasm clear of DNA, cells use the DNase Trex1 and the autophagy machinery (Stetson et al, 2008; Liang et al, 2014). There are also reports demonstrating a role for components of the autophagy pathway in negative regulation of STING-dependent signaling (Saitoh et al, 2009; Konno et al, 2013). It is known that STING is turned over following stimulation of signaling (Konno et al, 2013), and although this is believed to be central for negative control of the pathway, there is limited knowledge on the events that govern STING turnover. Activation of the cGAS-STING pathway occurs on membranous surfaces. STING is an endoplasmic reticulum (ER)-resident protein in the resting state (Paludan & Bowie, 2013). Following stimulation, STING traffics to the Golgi and ER-Golgi intermediate compartments (ERGIC) where TBK1 is recruited and the STING signalsome is activated including phosphorylation of STING and IRF3 (Saitoh et al, 2009; Dobbs et al, 2015; Liu et al, 2015). Eventually, STING can be found in speck-like structures (Ishikawa et al, 2009), which remain uncharacterized with respect to their nature and also potential roles in termination of STING signaling activity. It has been reported that these structures co-localize with autophagosome components, but previous studies found the formed membranes to be single bilayer vesicles, and not double bilayer membranes, as in autophagosomes (Saitoh et al, 2009). Recently, it was reported that STING mediates autophagy of the ER to release organelle stress, thus promoting antibacterial immunity and survival after infection (Moretti et al, 2017). While autophagy can be non-selective, there are receptors that can bind specific molecules and target them for autophagosomes (Levine et al, 2011). One important class of selective autophagy receptors bind and target ubiquitinated proteins to autophagosomes by interaction with LC3 through their LC3-binding region. Since ubiquitin is used extensively in activation of signals by pattern recognition receptors there is potential for crosstalk between the signals that stimulate immune signaling and the autophagy machinery (Gack et al, 2007; Damgaard et al, 2012; Wang et al, 2014). Here, we report that the ubiquitin-binding selective autophagy receptor p62/SQSTM1 is essential for DNA- and cGAMP-stimulated degradation of STING. STING in turn is ubiquitinated through K63 linkage and recruited to p62-positive compartments. This proceeds through a pathway where TBK1 phosphorylates p62 in an IRF3-dependent but transcription independent manner, which increases ubiquitin-binding affinity of p62. Consequently, STING is not degraded in p62-deficient cells, which produce elevated levels of type I IFN and IFN-stimulated genes (ISG)s. Thus, p62 is essential for targeting of STING for autophagosomal degradation following stimulation of the cGAS-STING pathway. Results DNA-stimulated turnover of STING occurs through autophagy Cytosolic DNA leads to degradation of STING (Konno et al, 2013). This was observed in both murine and human cells following stimulation with dsDNA or cGAMP (Figs 1A and EV1A–C) and was also observed in mice stimulated with the STING agonist DMXAA (Fig 1B). Defects in proteins of different branches of the autophagy machinery have been reported to lead to elevated innate response to DNA (Fig EV1D), but this has been suggested to occur independently of autophagy (Saitoh et al, 2009; Konno et al, 2013). When we tested the impact of another autophagy-related protein, ATG3, we found that Atg3−/− cells produced significantly elevated levels of the ISG CXCL10 after stimulation through the STING pathway as compared to wild-type (WT) cells (Fig 1C). Previous studies had failed to find DNA-stimulated formation of double-membrane autophagosomes (Saitoh et al, 2009). However, under our experimental conditions, we found that DNA stimulation led to increased cytoplasmic levels of (i) electron-dense vesicles resembling endosomes or lysosomes with single outer membrane (Figs 1D–F and EV1E), and (ii) double-membrane vesicles containing membranous structures (Figs 1G and H, and EV1F). Consistent with previous reports (Saitoh et al, 2009; Konno et al, 2013), treatment with DNA- or cGAMP-stimulated dephosphorylation at serine 555 of ULK1, which is an important component of the autophagy initiation complex (Figs 1I–K, and EV1G and H), as well as LC3-I to LC3-II conversion, and recruitment of LC3 to STING foci (Figs 1A and EV1I). Moreover, time-lapse fluorescence microscopy revealed that LC3 was recruited to the STING-positive vesicles that accumulated after stimulation with DMXAA (Figs 1L and EV1J; Movie EV1). Finally, we examined the effect of inhibition of autophagy on DNA-stimulated STING degradation. Importantly, STING was not degraded after DNA stimulation in Atg3−/− cells (Fig 1M), and this process was significantly delayed in Ulk1/2−/− cells in agreement with previous reports (Fig 1J; Hu et al, 2016; Konno et al, 2013). Moreover, inhibition of autophagy at different steps by 3-methyladenine and Bafilomycin A1, but not inhibition of the proteasome, impaired stimulation-induced degradation of STING (Fig EV1K–M). Bafilomycin A1 treatment furthermore augmented DNA-induced CXCL10 production (Fig EV1N). Collectively, stimulation of the cGAS-STING pathway leads to activation of several markers of autophagy and inhibition of autophagy impairs DNA-stimulated degradation of STING. Figure 1. DNA-stimulated turnover of STING occurs through autophagy A. WT and Stinggt/gt MEFs were treated with dsDNA (4 μg/ml) for the indicated time interval, and lysates were immunoblotted for STING, LC3, and β-actin. B. cGas−/− mice were treated with DMXAA (500 mg/mouse) for the indicated time interval, and spleen lysates were immunoblotted for STING and β-actin. C. WT and Atg3−/− MEFs were treated with dsDNA (4 μg/ml) or 2′3′ cGAMP (4 μg/ml) for 18 h, and supernatants were harvested and analyzed for levels of CXCL10. D, E. Electron microscopy of glutaraldehyde-fixed cells treated with Lipofectamine (mock) or transfected with dsDNA (4 μg/ml) for 8 h. Panel (E) represents 10× magnifications of selected areas of the DNA-stimulated cells. Red arrowheads highlight electron-dense vesicles in DNA-stimulated cells. Scale bars: 2 μm (D); 250 nm (E). F. Stereological analysis of data shown in panels (D, E). The graph is based on quantification of at least 35 cell profiles per group. G. Electron microscopy of glutaraldehyde-fixed cells transfected with dsDNA (4 μg/ml) for 8 h. Red arrowheads highlight double-membrane characteristic for autophagosomes. Scale bar: 250 nm. H. Stereological analysis of data shown in panel (G). The graph is based on quantification of at least 35 cell profiles per group. I. WT and Ulk1/2−/− MEFs were treated with dsDNA (4 μg/ml) for the indicated time interval, and lysates were immunoblotted for pULK1 S555, ULK, STING, and β-actin. J. Control, cGAS KO, and STING KO THP1 cells were treated with dsDNA (4 μg/ml) for the indicated time interval, and lysates were immunoblotted for cGAS, STING, pTBK1, pULK1, and β-actin. K. WT and Ulk1/2−/− MEFs were treated with dsDNA (4 μg/ml) or 2′3′ cGAMP (4 μg/ml) 18 h, and supernatants were harvested and analyzed for levels of CXCL10. L. MEFs were transiently transfected with STING-mCherry and LC3-EGFP, treated with DMXAA (100 μg/ml), and imaged live with wide-field microscopy. Representative image sequence of STING-mCherry and LC3-EGFP is shown as inverted contrast. Arrowheads point to LC3-EGFP recruitment to STING-mCherry compartments. Scale bar: 5 μm. M. WT and Atg3−/− MEFs were treated with dsDNA (4 μg/ml) for the indicated time interval, and lysates were immunoblotted for STING, pTBK1, LC3, and β-actin. Data information: The data in panels (C, F, H, K) are presented as means ± s.d. *0.01 < P < 0.05; **0.001 < P < 0.005; ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. DNA and cGAMP stimulate STING degradation in a range of cell types by autophagy A, B. WT MEFs were treated with 2′3′ cGAMP (4 μg/ml) or DMXAA (100 μg/ml) for the indicated time interval, and lysates were immunoblotted for STING and β-actin. C. THP1 cells were treated with dsDNA (4 μg/ml) for the indicated time interval, and lysates were immunoblotted for STING, LC3, and β-actin. D. Illustration of the canonical autophagy pathway. 3MA, 3-methyladenine. BafA1, Bafilomycin A1. E, F. Electron microscopy of sections from glutaraldehyde-fixed cells transfected with dsDNA (4 μg/ml) for 8 h. Panel (E) shows DNA-stimulated lysosome-like vesicle, and panel (F) shows two images of vesicular structures enclosed by double membrane (autophagosome) or single membrane (autophagolysosome). Scale bar: 250 nm. Arrowheads: Vesicle outer membranes. G. Control, cGAS KO, and STING KO THP1 cells were treated with 2′3′ cGAMP (4 μg/ml) for the indicated time interval, and lysates were immunoblotted for cGAS, STING, pULK1 and β-actin. H. WT and Ulk1/2−/− MEFs were treated with dsDNA (4 μg/ml) for the indicated time interval, and lysates were immunoblotted for pTBK1 and β-actin. I. WT MEFs were treated with dsDNA (4 μg/ml) for 8 h, and stained with DAPI, anti-STING, and anti-LC3. Scale bar, 5 μm. J. MEFs were transiently transfected with STING-mCherry and LC3-EGFP, treated with DMXAA (100 μg/ml) and imaged live with wide-field microscopy. Upper panel, representative image sequence of a cell at different time points after stimulation. Lower panel, representative image sequence of the boxed regions. STING-mCherry and LC3-EGFP are shown as inverted contrast. Time is indicated as the time in minutes after the start of image acquisition. Arrowheads point to LC3-EGFP recruitment to STING-mCherry compartments. Scale bars, 5 μm. K–M. THP1 cells were treated with MG132 (20 mM), 3MA (5 mM) or Baf1A (10 μM) 1 h prior to stimulation with dsDNA (4 μg/ml). Lysates were isolated 9 h post-stimulation and immunoblotted for STING and β-actin. N. Supernatants from the cells treated as in panel (M) were isolated and levels of CXCL10 were determined by ELISA. Data are shown in means of triplicates ± s.d. Download figure Download PowerPoint The selective autophagy receptor p62/SQSTM1 exerts negative control of the cGAS-STING pathway Ubiquitination is involved in both proteasomal and autophagosomal degradation of proteins, and STING is ubiquitinated at specific residues to promote both activation and negative regulation (Zhong et al, 2009; Tsuchida et al, 2010; Zhang et al, 2012; Wang et al, 2014; Ni et al, 2017). To examine whether specific types of ubiquitin-linkage accumulated around STING after DNA treatment, stimulated cells were stained with antibodies specific for K63- and K48-linked ubiquitin chains. The well-described trafficking of STING to specific specks was clearly observed, and there was a strong co-localization with total ubiquitin, K63-linked ubiquitin, and to a modest extent with K48-linked ubiquitin (Figs 2A and B, and EV2A, Appendix Table S1). The observed positive STING signal by microscopy 8 h after stimulation is in contract to the very low STING levels observed at the same time points by Western blotting. This discrepancy is currently not explained. One contributing factor could be that STING degradation products in autophagosomes may be detected by microcopy but not Western blotting. Figure 2. The selective autophagy receptor p62/SQSTM1 exerts negative control of the cGAS-STING pathway A, B. WT MEFs were stimulated with dsDNA (4 μg/ml). The cells were fixed 8 h later and stained with antibodies against total ubiquitin or K63-linked ubiquitin. Scale bars: 5 μm. C. THP1 cells were stimulated with dsDNA (4 μg/ml) for 8 h, and lysates were immunoblotted for p62, NBR1, OPTN, and NDP52. D–G. WT MEFs were stimulated with dsDNA (4 μg/ml). The cells were fixed 8 h later and stained with antibodies against total p62, OPTN, NDP52, or NBR1. Scale bars: 5 μm. H, I. WT and p62−/− MEFs were stimulated with dsDNA (4 μg/ml) or 2′3′ cGAMP (4 μg/ml). Total RNA and supernatants were harvested 6 and 18 h later, respectively, and levels of Ifnb and CXCL10 were measured. J. The RNA analyzed in panel (H) was also subjected to a wider examination of ISGs by Fluidigm. The data are presented as a heatmap with each color representing the mean of triplicate measurements. K. Control and p62 KO THP1 cells were stimulated with dsDNA (4 μg/ml) or 2′3′ cGAMP (4 μg/ml). Supernatants were harvested 18 h later, and levels of CXCL10 were measured. L, M. THP1 cells were left untreated or stimulated with dsDNA (4 μg/ml) for 4 h in the presence or absence of 3MA (5 mM). p62 was immunoprecipitated from whole-cell lysates, and levels of STING, TBK1, and p62 in the precipitates were evaluated by immunoblotting. N. THP1 cells were stimulated with dsDNA (4 μg/ml) or 2′3′ cGAMP (4 μg/ml). The cells were fixed 8 h later and stained with antibodies against p62, K63-linked ubiquitin, and STING. Scale bar: 5 μm. O. THP1 cells were stimulated with dsDNA (4 μg/ml) for 8 h and stained with antibodies against STING, p62, and beclin-1. Scale bar: 5 μm. Data information: The data in panels (H, I, K) are presented as means of 3–5 replicates ± s.d. **0.001 < P < 0.005; ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. p62/SQSTM1 is turned over after DNA stimulation and regulates the cGAS-STING pathway A. WT MEFs were stimulated with dsDNA (4 μg/ml). The cells were fixed 8 h later and stained with antibodies against K48-linked ubiquitin. Scale bar: 5 μm. Arrow: area of clear colocalization in the stainings. B. Illustration of the ubiquitin-binding selective autophagy receptors. C, D. WT, Nbr1−/−, and Optn−/− MEFs were stimulated with dsDNA (4 μg/ml) or 2′3′ cGAMP (4 μg/ml). Supernatants were harvested 18 h later, and levels of CXCL10 were measured by ELISA. E. Lysates from DNA-stimulated WT and Optn−/− MEFs were immunoblotted for the indicated proteins. F. WT and Hdac6−/− MEFs were stimulated with dsDNA (4 μg/ml). Total RNA was harvested 6 h later, and levels of Ifnb mRNA were measured by RT–qPCR. G. Illustration of the localization of the target regions in the p62 gene of the two gRNAs used to generate p62 KO THP1 cells. H. Lysates from control THP1 and two THP1-derived p62-deficient cell lines generate with two different gRNAs were immunoblotted for p62, STING, TBK1, IRF3, and β-actin. I. Control and p62 KO THP1 cells (gRNA#2) were stimulated with dsDNA (4 μg/ml). Supernatants were harvested 18 h later, and levels of CXCL10 were measured. J, K. Human foreskin fibroblasts treated with control and p62-specific gRNA were stimulated with dsDNA (4 μg/ml) or 2′3′ cGAMP (4 μg/ml). Total RNA and supernatants were harvested 6 and 18 h later, respectively, and levels of IFNB and CXCL10 were measured. Western blots for p62 are shown to the right. L, M. WT and p62-deficient MEFs and THP1 cells were stimulated with dsDNA (4 μg/ml). Supernatants were harvested at the indicated time points, and levels of CXCL10 were determined by ELISA. N. THP1 cells were stimulated with dsDNA (4 μg/ml) for 8 h and stained with antibodies against STING, p62, and cGAS. Scale bar: 5 μm. Arrow: area of clear colocalization in the stainings. Data information: (C, D, F, I–M) Data are presented as means of 3–5 replicates ± s.d. **0.001 < P < 0.005; ***P < 0.001. Download figure Download PowerPoint A family of receptors is known to selectively target ubiquitinated proteins for autophagy, through their molecular architecture with both an LC3-interacting region and a ubiquitin-binding domain (Fig EV2B). Four of these selective autophagy receptors are p62/SQSTM1, NBR1, NDP52, and optineurin (OPTN). All four receptors were expressed in THP1 cells, and p62 and OPTN were also degraded in response to DNA (Fig 2C). In addition, we observed a pronounced relocalization of p62, and to a lesser extent also OPTN, NBR1 and NDP52, to the same areas as STING upon stimulation (Fig 2D–G, Appendix Table S1). Thus, p62 was strongly localized to the same areas as STING after DNA stimulation, and p62 and OPTN were degraded following DNA stimulation. In order to examine the role of p62 in regulation of DNA-stimulated IFN responses, we compared expression of IFNβ and CXCL10 in WT versus p62−/− cells. Interestingly, MEFs lacking p62 induced significantly elevated levels of IFNβ and ISGs after stimulation with DNA or cGAMP (Fig 2H–J). Although NBR1 and OPTN also co-localized with STING, cells deficient in either autophagy receptor did not exhibit elevated expression of CXCL10 after stimulation with DNA or cGAMP (Fig EV2C and D). Optn−/− MEFs even exhibited reduced signaling and CXCL10 expression after stimulation of STING (Fig E
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