STING promotes senescence, apoptosis, and extracellular matrix degradation in osteoarthritis via the NF-κB signaling pathway

Abstract

Damaged deoxyribonucleic acid (DNA) is a primary pathologic factor for osteoarthritis (OA); however, the mechanism by which DNA damage drives OA is unclear. Previous research demonstrated that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) participates in DNA damage response. As a result, the current study aimed at exploring the role STING, which is the major effector in the cGAS-STING signaling casacde, in OA progress in vitro, as well as in vivo. In this study, the expression of STING was evaluated in the human and mouse OA tissues, and in chondrocytes exposed to interleukin-1 beta (IL-1β). The influences of STING on the metabolism of the extracellular matrix (ECM), apoptosis, and senescence, were assessed in STING overexpressing and knocking-down chondrocytes. Moreover, the NF-κB-signaling casacde and its role in the regulatory effects of STING on ECM metabolism, apoptosis, and senescence were explored. The STING knockdown lentivirus was intra-articularly injected to evaluate its therapeutic impact on OA in mice in vivo. The results showed that the expression of STING was remarkably elevated in the human and mouse OA tissues and in chondrocytes exposed to IL-1β. Overexpression of STING promoted the expression of MMP13, as well as ADAMTS5, but suppressed the expression of Aggrecan, as well as Collagen II; it also enhanced apoptosis and senescence in chondrocytes exposed to and those untreated with IL-1β. The mechanistic study showed that STING activated NF-κB signaling cascade, whereas the blockage of NF-κB signaling attenuated STING-induced apoptosis and senescence, and ameliorated STING-induced ECM metabolism imbalance. In in vivo study, it was demonstrated that STING knockdown alleviated destabilization of the medial meniscus-induced OA development in mice. In conclusion, STING promotes OA by activating the NF-κB signaling cascade, whereas suppression of STING may provide a novel approach for OA therapy.

Fig. 1. The expression of STING is increased in osteoarthritic human and mouse articular cartilage.

A–B Representative H&E staining, Safranin O staining as well as immunofluorescence staining, immunohistochemistry staining in human articular cartilage from normal, as well as OA patients (bar: 50 μm). C–D Representative H&E staining, Safranin O staining, as well as immunofluorescence staining, immunohistochemical staining in mouse knee articular cartilage from sham, as well as DMM models (bar: 50 μm). E The protein expression of STING in chondrocytes originated from normal and degenerated specimens of human. F The protein expression of STING in chondrocytes originated from normal group and DMM (degenerated) specimens of mouse. All data are indicated as the mean ± SD (n = 5); **P < 0.01.

Fig. 2. The expression of STING is increased in IL-1β-triggered chondrocytes.

The protein expression of γh2ax, cGAS, STING in chondrocytes were explored by western blot and its quantification was detected by ImageJ. A–B The impact of different concentrations of treatment with IL-1β on mouse chondrocytes. The chondrocytes were exposed to 0, 10, 20, 30, 40 (ng/ml) IL-1β for 24 h. C–D The impact of different durations of treatment with IL-1β on mouse chondrocytes. The chondrocytes were incubated with IL-1β (10 ng/ml) for 0, 6, 12, 24 h. E Representative immunofluorescence staining of γh2ax in IL-1β (10 ng/ml, 24 h) induced chondrocytes (bar:10μm). All data were indicated as the mean ± SD (n = 5); *P < 0.05, **P < 0.01.

Fig. 3. STING promotes ECM degradation, senescence, and apoptosis in chondrocytes.

The mouse chondrocytes were pre-exposed to lv-STING or sh-STING and subsequently by IL-1β (10 ng/ml) treatment for 24 h. A Western blot, as well as its quantification revealed the level of STING, Aggrecan, and Collagen II, MMP13 and ADAMTS5 after exposure to IL-1β (10 ng/ml) or lv-STING. B Western blot along with its quantification revealed the level of STING Aggrecan, and Collagen II, MMP13 and ADAMTS5 after treatment with IL-1β or sh-STING. C Western blot along with its quantification revealed the level of STING, BAX and BCL-2, and Cyto-c, cleaved-caspase-3 and p16INK4a, and p21 after exposure to IL-1β or lv-STING. D Western blot along with its quantification revealed the level of STING, BAX, and BCL-2, and cleaved-caspase-3 and p16INK4a, and p21 after exposure to IL-1β or sh-STING. All data are indicated as mean ± SD (n = 5); *P < 0.05, **P < 0.01.

Fig. 4. STING induces ECM degradation via NF-κB activation.

The mouse chondrocytes were pre-exposed to lv-STING or sh-STING or si-P65 followed by exposure to IL-1β (10 ng/ml) for 24 h. A–B Western blot as well as its quantification revealed the phosphorylation level of P65 and iκb after exposure to IL-1β or lv-STING. C–D Western blot along with its quantification revealed the phosphorylation level P65, as well as iκb after exposure to IL-1β or sh-STING. E Immunofluorescence staining of P65 in chondrocytes (bar: 50 μm). F–G Western blot along with its quantification revealed that the siRNA-mediated P65 knockdown was successful. H–I Western blot along with its quantification revealed that the level of Collagen II, Aggrecan, MMP13, and ADAMTS5 as treated above. J–K Immunofluorescence staining of collagen II in chondrocytes as treated above (bar:50μm). All data were presented as mean ± SD (n = 5); **P < 0.01.

Fig. 5. STING induces chondrocyte senescence and apoptosis via NF-κB activation.

The mouse chondrocytes were pre-exposed to lv-STING or si-p65 followed by exposure to IL-1β (10 ng/ml) for 24 h. A–C Western blot and its quantification of the level of BAX, BCL-2, Cyto-c, cleaved-caspase-3, p16INK4a and p21. D–F TUNEL staining assay was conducted on the chondrocytes, as treated above (bar: 100 μm). E–G SA-β-gal staining assay was carried out on chondrocytes, as shown (bar: 50 μm). All data were indicated as mean ± SD (n = 5); **P < 0.01.

Fig. 6. STING knockdown ameliorates osteoarthritis in vivo.

The osteoarthritis mouse model was created by surgical destabilization of the medial meniscus (DMM), after 8-week time period, the pathology of OA was assessed by X-ray or stained with H&E, as well as Safranin O. A Digital X-ray image of mouse knee joints from different experimental groups. B Representative immunohistochemistry of STING, S-O staining of the cartilage and synovitis in the four groups at eight weeks post-surgery (bar: 50 or 25 μm). C Quantitative analysis of immunohistochemistry of STING was detected by image J. D–E The OARIS scores of the cartilage and the scores of the synovitis in the four groups. All data were presented as mean ± SD (n = 15), **P < 0.01.

Fig. 7. STING knockdown ameliorates the degradation of the ECM, senescence, and apoptosis by inhibiting NF-κB in vivo.

A–B The expression of p-P65, p16INK4a, P21, and collagen II were evaluated by immunohistochemistry in mouse cartilage (bar: 50 μm). C–D TUNEL staining assay in mouse cartilage (bar: 50 μm). All data were presented as mean ± SD (n = 15); **P < 0.01.

Fig. 8

Schematic illustration of effect of cGAS-STING in osteoarthritis development.