Brachyury promotes extracellular matrix synthesis through transcriptional regulation of Smad3 in nucleus pulposus.

Metabolic dysfunction of the extracellular matrix (ECM) is one of the primary causes of intervertebral disc degeneration (IVDD). Previous studies have demonstrated that the transcription factor Brachyury has the potential to promote the synthesis of collagen II and aggrecan, while the specific mechanism is still unknown. In this study, we used a lipopolysaccharide (LPS)-induced model of nucleus pulposus cell (NPC) degeneration and a rat acupuncture IVDD model to elucidate the precise mechanism through which affects collagen II and aggrecan synthesis in vitro and in vivo. First, we confirmed Bry expression decreased in degenerated human nucleus pulposus (NP) cells (NPCs). Knockdown of exacerbated the decrease in collagen II and aggrecan expression in the lipopolysaccharide (LPS)-induced NPCs degeneration in vitro model. Bioinformatic analysis indicated that may participate in the regulatory pathway of ECM synthesis regulated by Bry. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays demonstrated that enhances the transcription of by interacting with a specific motif on the promoter region. In addition, Western blot and reverse transcription-qPCR assays demonstrated that positively regulates the expression of aggrecan and collagen II in NPCs. The following rescue experiments revealed that -mediated regulation of ECM synthesis is partially dependent on Smad3 phosphorylation. Finally, the findings from the in vivo rat acupuncture-induced IVDD model were consistent with those obtained from in vitro assays. In conclusion, this study reveals that positively regulates the synthesis of collagen II and aggrecan in NP through transcriptional activation of Smad3. Mechanically, in the nucleus, enhances the transcription of , leading to increased expression of Smad3 protein levels; in the cytoplasm, elevated substrate levels further lead to an increase in the phosphorylation of Smad3, thereby regulating collagen II and aggrecan expression. Further in vivo experiments provide additional evidence that can alleviate IVDD through this mechanism.