Background: While Nitric oxide (NO) and Notch signaling pathways are implicated in osteoarthritis (OA) progression, their functional interplay remains largely unexplored. We hypothesized that Gucy1a3 (sGCα 1) functions as a non-canonical soluble sensor, bridging nitrosative stress to pathological Notch activation. Methods: To test the hypothesis that Gucy1a3 mediates NO-Notch crosstalk, we used both sodium nitroprusside (SNP)-stimulated chondrocytes and a rat medial meniscus resection model. Mechanistic hierarchy was analyzed via Gucy1a3 siRNA knockdown and the Notch inhibitor DAPT. Additionally, the capacity of DAPT to modulate chondrogenic lineage commitment was evaluated in rat bone marrow mesenchymal stem cells (BMSCs). Results: DAPT reversed SNP-induced catabolism in chondrocytes, suppressing Notch components (Notch1, Jagged1) and inflammatory markers (iNOS, MMP13) while restoring anabolic gene expression. Crucially, DAPT suppressed Gucy1a3 expression, breaking a reciprocal positive feedback loop (NO-Gucy1a3-Notch). Although Gucy1a3 knockdown phenocopied the anti-inflammatory signature of DAPT, DAPT uniquely activated the chondrogenic differentiation of BMSCs. In preclinical models, intra-articular delivery of DAPT effectively safeguarded joint homeostasis by reducing subchondral bone deterioration and decreasing cartilage degeneration. Conclusion: This study identifies Gucy1a3 as a mediator that transduces inflammatory nitric oxide signals to activate Notch. DAPT exerts therapeutic effects by inhibiting Gucy1a3-mediated catabolism in chondrocytes and promoting the differentiation of BMSCs. Although these findings are currently limited to in vitro and rodent models, targeting this signaling axis offers a potential strategy for developing disease-modifying osteoarthritis drugs. The illustration compares pathological and therapeutic pathways in cartilage treatment. On the left, the pathological pathway shows activation of Gucy1a3 by nitric oxide, leading to increased catabolic markers (iNOS, MMP13) and decreased anabolic markers (Aggrecan, Col2a1) in the nucleus. On the right, the therapeutic pathway involves DAPT and siRNA inhibiting Gucy1a3, resulting in increased anabolic markers and decreased catabolic markers. Bone marrow-derived stem cells (BMSCs) treated with DAPT form cartilage globules. Below, two knee joint illustrations compare osteoarthritis (OA) untreated and treated with DAPT, showing improved cartilage in the treated joint.Illustration of pathological vs. therapeutic pathways in cartilage treatment using DAPT and siRNA. Keywords: osteoarthritis, Gucy1a3, notch signaling, DAPT, cartilage
Zheng et al. (Fri,) studied this question.