Double-stranded DNA Induces STING Degradation And Regulatory Mechnism Study

The stimulator of interferon gene (STING) protein has emerged as a critical signal transduction protein of the double-stranded DNA (dsDNA)-induced innate immune signaling pathway. It mediates the production of type IIFN and other cytokines such as IL-6 and IL-1β, which exert antiviral and antibacterial activities. As an important signal transduction protein, the STING protein level is directly associated with the intensity of the dsDNA-induced innate immune response. Previous studies have revealed that a variety of cellular molecular mechanisms can regulate STING protein level through ubiquitin/proteasome-mediated protein degradation with consequent effect on the intensity of the innate immune response. Based on the previous findings, we attempted to further investigate the mechanisms responsible for the regulation of STING protein stability for further improving our understanding of the dsDNA-induced innate immune.In the present study, we first revealed that the transfection of plasmid DNA, used as a representative of dsDNA, significantly reduced the expression of STING protein in MRC-5 cells in both time-dependent and dose-dependent manners. In addition to plasmid DNA, other dsDNA, such as PCR-amplified dsDNA fragment, human genomic DNA, and different viral genomic DNA, induced a similar decrease in STING protein level, demonstrating that the decrease in STING protein level is a common event induced by dsDNA. The results from detecting the length of DNA fragments showed that the DNA fragment shorter than 1000bp was not able to induce the decrease of STING protein level.To study the biological effects of the STING protein degradation, we studied the relationship between STING protein and retinoic acid inducible gene I (RIG-I), IL-6 and IFN-β. First, we analyzed the expression of these molecules following the reduction of STING protein expression. It was found that the silencing of STING expression by it specific siRNA (siSTING) caused a significant reduction of RIG-I expression, thus for the first time showing that RIG-I can not only serve as RNA or DNA receptors, but also be a downstream molecule of the STING protein. With the previous findings that the down-regulation of STING can reduce the expression of both IL-6 and IFN-β, these studies suggested that these three molecules are downstream of the STING signaling. Second, it was attempted to determine whether RIG-I, IL-6 and IFN-β participated in the regulation of STING protein degradation. The results showed that:1) The down-regulation of RIG-I by its specific siRNA (siRIG-I) partially elevated the STING protein level; 2) The treatment with anti-IL-6 antibody caused a slight increase in STING protein level, suggesting that the dsDNA-induced IL-6 also contributes to the STING protein degradation; 3) Addition of IFN-β, however, did not reduce STING protein level, suggesting that IFN-β alone may be not able to regulate STING protein stability. Finally, the relationship between the effect of the combined expression of RIG-I, IL-6, IFN-β and the regulation of STING protein stability was investigated. The results showed that IFN-β treatment significantly induced RIG-I expression, suggesting that RIG-I is downstream of the IFN-β signaling. Since the treatment with IFN-β failed to induce STING protein degradation, it was suggested that the elevation of RJG-I alone could not initiate the STING degradation. However, co-treatment with siRIG-1 and IL-6 antibody did show an additive effect in the inhibition of STING degradation. Given that the inhibition of the STING degradation by the co-treatment with RIG-I siRNA and IL-6 antibody was incomplete, it was further suggested that, in addition to RIG-I and IL-6, other molecules were also involved in the STING degradation. In summary, the STING protein plays a very important role in mediating the dsDNA-induced expression of RIG-I, IL-6 and IFN-β. However, in order to limit the inducted effect by the STING protein with the consequent restriction on the intensity of the dsDNA-induced innate immune response, the sustained increase in RIG-I, IL-6, IFN-β and perhaps other molecules may send the feedback signaling leading to the degradation of STING protein.To further understand the mechanism(s) responsible for the STING protein degradation, Bortezomib, a proteasome inhibitor, was used to treat the cells during dsDNA transfection. Through an immunoprecipitation assay, it was shown that the STING degradation happened via the ubiquitin-proteasome degradation system. However, the RNF5 protein, which was reported previously as the E3 ligase for the STING protein degradation, was ruled out in testing system involving in the STING degradation. To identify the amino acid residues served as the ubiquitination sites for the STING degradation, all lysine residues of the protein were mutated in vitro. But none of the point mutation showed any effect on the reversal of STING protein degradation, suggesting that the STING protein degradation may occur via nonconventional ubiquitination amino acid residues. In addition, specific downregulation of interferon stimulated genes 15(ISG15) protein level by its specific siRNA was found to be able to further promote the degradation of STING protein, suggesting that the STING protein stability is an outcome of a dynamic balance between ubiquitination and ISGylation.In order to determine the existence of the negative feedback mechanism involved in the STING protein degradation also in other cells, the human diploid cells 2BS and KMB17, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs), tumor cell lines NCI-H358、 Hela and NCI-H1703, and the immortalized cell lines HEK293 and BEAS were examined similarly for the dsDNA-induced STING degradation. Except for HEK293 and NCI-H1703, the same finding in the STING protein degradation accompanied by the increase in IL-6 and RIG-I were detected in all these cells, thus suggesting that the mechanism for regulating the STING protein stability is a common mechanism, even though some tumor cell lines or immortalized cell lines have lost this mechanism.The present study revealed a negative feedback mechanism existing in various cells for keeping the excessive immune response induced by dsDNA in tight control. With several novel findings, this study further improved our understanding of the dsDNA-associated innate immune responses.