STING, the Endoplasmic Reticulum, and Mitochondria: Is Three a Crowd or a Conversation?

Abstract

The anti-viral pattern recognition receptor STING and its partnering cytosolic DNA sensor cGAS have been increasingly recognized to respond to self DNA in multiple pathologic settings including cancer and autoimmune disease. Endogenous DNA sources that trigger STING include damaged nuclear DNA in micronuclei and mitochondrial DNA (mtDNA). STING resides in the endoplasmic reticulum (ER), and particularly in the ER-mitochondria associated membranes. This unique location renders STING well poised to respond to intracellular organelle stress. Whereas the pathways linking mtDNA and STING have been addressed recently, the mechanisms governing ER stress and STING interaction remain more opaque. The ER and mitochondria share a close anatomic and functional relationship, with mutual production of, and inter-organelle communication via calcium and reactive oxygen species (ROS). This interdependent relationship has potential to both generate the essential ligands for STING activation and to regulate its activity. Herein, we review the interactions between STING and mitochondria, STING and ER, ER and mitochondria (vis-à-vis calcium and ROS), and the evidence for 3-way communication.

Keywords: STING; cGAS; endoplasmic reticulum; mitochondria; reactive oxygen species; unfolded protein response.

Figure 1

STING stimulation by stressed organelles: an interactive triad. STING plays a critical role in preserving health but also mediates disease, even in the absence of infectious triggers. Mitochondrial DNA (red lines) has recently emerged as a trigger of STING activation. The endogenous ligand mediating the ER-STING reciprocal relationship is not clear. The endoplasmic reticulum (ER) and mitochondria share a very close anatomic and functional relationship, and together modulate homeostatic and pathologic levels of intracellular calcium (Ca²⁺) and reactive oxygen species (ROS). This relationship may generate the “missing ligand” for ER stress-mediated STING activation via mitochondrial DNA release.

Figure 2

cGAS and STING activation. Cytosolic dsDNA from viruses, mitochondria or nucleoids formed during nuclear breakdown bind cGAS, triggering its catalytic formation of 2’3’ cGAMP. 2’3’ cGAMP serves as a ligand for STING, which resides in the ER with its ligand binding domain (LBD) facing the cytosol. TM=transmembrane domain. Bacterial cyclic-di-nucleotides, such as cyclic-di-AMP, cyclic-di-GMP and 3’3’ cGAMP also bind STING. Upon ligand binding, the cytosolic domains of STING close over the di-nucleotide ligand and rotate 180 degrees, enabling lateral stacking. STING translocates to the Golgi where it oligomerizes. This oligomerization enhances trans-phosphorylation of the STING CTT (C terminal tail)-associated TBK1 family kinases. TBK1 has a scaffold and dimerization domain (SDD), ubiquitin like domain (Ub) and Kinase domain (KD). Activated TBK1 phosphorylates the STING CTT, enabling recruitment and subsequent phosphorylation of IRF3. TBK1 family kinases also activate signaling pathways leading to NF-κB nuclear translocation. STING activation has diverse immune stimulatory outputs including pro-inflammatory cytokine responses (via NF-κB), interferon responses (via IRF3), apoptosis and autophagy. STING/TBK1 structural cartoon adapted from (51).

Figure 3

Concept map of STING in sterile pathology. Irradiation, cancer, aging/senescence and infection can drive genotoxic stress, resulting in the generation of micronuclei. Nuclease mutations, deficiencies, and mitochondrial DNA release lead to increased cytosolic dsDNA. These immediate drivers of cytosolic dsDNA are in red boxes. STING aberrantly activated through these processes, as well as STING mutations and altered STING regulation (blue boxes) all result in pathologic disease states. Excess STING-dependent IFN and inflammatory cytokines contribute to pathology (green boxes) in Type I interferonopathies, autoimmunity and post-MI (Myocardial Infarction). However the effects of STING on other types of pathologies (Cancer, Diabetes) can vary (yellow boxes) depending upon the specific situation.

Figure 4

Connection between oxidized mtDNA and Lupus. Stimulation of IFN-treated neutrophils or neutrophils from lupus patients with anti-RNP immune complexes can lead to release of oxidized (ox) mtDNA by multiple mechanisms: 1) Stimulation of NETosis, with extrusion of DNA containing oxidized mtDNA. 2) Increased mitochondrial ROS leads to membrane translocation and extrusion of oxidized mtDNA into the extracellular milieu. 3) Anti-RNP and type I IFN decrease the levels of cyclic AMP (cAMP), a second messenger required for activation of protein kinase A (PKA), which normally phosphorylates TFAM, enabling its release from mtDNA. When TFAM is released, the mtDNA can then go to the lysosome for degradation. If PKA is inhibited, TFAM remains associated with mtDNA in nucleoids that accumulate in mitochondria and then are released from the neutrophils through unclear mechanisms. Extracellular oxidized mtDNA is sensed by monocytes in a STING-dependent manner and internalized by pDC via RAGE receptors. Downstream of RAGE, the IFN-generating sensor in pDC is unclear, although both oxidized and non-oxidized mtDNA stimulation of pDC is TLR9-dependent (139). The abundance of anti-mtDNA antibodies in lupus and correlation with disease support the critical involvement of these mechanisms in disease pathogenesis (, , and 140).

Figure 5

Unfolded Protein Response (UPR), STING and autophagy. When cellular insults or protein production demands compromise ER function, the ER initiates the UPR. Misfolding proteins bind the chaperone BiP, releasing it from three stress sensors, IRE1 (blue), ATF6 (green) and PERK (red). IRE1 is a bifunctional kinase/endonuclease that initiates JNK-dependent signaling and excises a 26bp stretch from the XBP1 mRNA, removing a premature stop codon via frameshift mutation. IRE1 also decreases ER load through more promiscuous endonuclease activity (RIDD). Upon release of BiP, ATF6 translocates to the Golgi, where S1 and S2 proteases generate an active transcription factor. PERK kinase phosphorylates eIF2α, resulting in global translational attenuation apart from select mRNAs such as ATF4. ATF4 promotes transcription of the pro-apoptotic transcription factor CHOP and Ero1α oxidoreductase. PERK also leads to nuclear factor erythroid 2–related factor 2 (Nrf2) nuclear translocation and resulting anti-oxidant responses. The UPR promotes STING activity and STING increases the UPR (right green arrows). This STING-dependent increase in UPR also enhances autophagy of ER components (“ER-phagy”, left side), which can limit ER stress responses. Many questions remain regarding the mechanistic details connecting STING, UPR and ERphagy.

Figure 6

Reciprocal effects of calcium on STING activity and STING on calcium homeostasis. Increases in cytosolic calcium (Ca²⁺) enhances STING activity through multiple mechanisms: 1) calcium directly binds STING dimers, promoting cyclic-di-nucleotide signaling, 2) increased cytosolic calcium enhances mitochondrial DNA extrusion (thus triggering cGAS), and 3) calcium stimulated calmodulin activates CAMKII, which phosphorylated AMPK, which then inhibits ULK1, a STING inhibitor. SERCA pump inhibitors Thapsigargin (Tpg), infection and oxygen glucose deprivation (OGD) increase cytosolic calcium, thereby stimulating STING. The mechanisms underlying these observations are not yet established. Too much calcium (as in ionomycin treatment) inhibits STING activity. On the right, STING stimulates IP3R-dependent calcium release, a process that may be counteracted by SERCA activity. In its inactive state, STING sequesters STIM1 in the ER, preventing extracellular calcium entry. STIM1 reciprocally “tethers” STING to the ER, inhibiting its activity.

Figure 7

ER-mitochondria connections at the ER mitochondria-associated membranes (MAMs). Mitochondria are closely associated with ER membranes through multiple sets of molecular bridges, including the mitofusins (Mfsn) that regulate mitochondria fission/fusion, the inositol triphosphate receptor (IP3R) calcium channel and non-selective voltage-dependent anion channel (VDAC) stabilized by GRP75, and Vesicle APC-Binding Protein (VAPB) and protein tyrosine phosphatase-interacting protein 51 (PTIP51), which also regulate calcium flux. ER stress sensors inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK) and folding chaperones (e.g. GRP78/BiP) congregate at the ER mitochondria- associated membranes (MAMs). STING is also enriched at the MAMs. In the resting state, STING associates with STIM1. ER calcium is primarily regulated by three types of calcium channel: Sarcoplasma/ER calcium ATPase (SERCA), which pumps calcium (Ca²⁺) into the ER, and the IP3Rs and ryanodine receptors (RyR), which release ER calcium. Mitochondrial respiration and the action of the electron transport chain (ETC) generate ROS. Protein folding is the primary source of ROS generation in the ER. PERK indirectly induces (dashed arrow) Endoplasmic Reticulum Oxidoreductase 1 Alpha (Ero1α) expression, which is one of the primary sources of ER ROS. ROS decrease ER calcium by inhibiting SERCA and activating IP3R and RyR. ROS also stimulate the translocation of Stromal interaction molecule 1 (STIM1) from ER to plasma membrane, where it interacts with Calcium release-activated calcium channel protein 1 (Orai1) to enable store operated calcium entry (SOC). SOC stimulates NADPH oxidase, generating a positive feedback loop. At the mitochondria, too much calcium and ROS stimulate Bak/Bax mediated release of cytochrome c and extrusion of mitochondrial DNA (mtDNA). The mtDNA stimulates cGAS production of 2’3’-cGAMP, an activating ligand for STING. Calcium regulating molecules are in green, apoptosis in solid yellow, and UPR-associated molecules as in Figure 5.

Figure 8

Different outcomes of PERK activation in T cells vs. MDSC and STING input. In T cells, PERK and ERO1α increase ROS production, leading to mitochondrial dysfunction, increased exhaustion and lower IFNγ production, rendering these PD1+ T cells less adept at fighting tumors. ROS-stimulated XBP1 also decreases T cell activation. Although STING activation could make matters worse by increasing UPR induction and PDL1 expression, many studies indicate a positive role for STING and type I IFN in pDC-dependent CD8 T cell activation and anti-tumor activities, suggesting a balance of effects. On the other hand, in MDSC, PERK-stimulated Nrf2 activity predominates. Nrf2 prevents mitochondrial ROS and dysfunction. Mitochondrial ROS also leads to dsDNA extrusion and STING activation, which further inhibit MDSC via Type I IFN signaling. MDSC promote tumor progression.