DDRGK1-mediated UFMylation system suppresses STAT3 phosphorylation to alleviate osteoarthritis

The phosphorylation of Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in regulating inflammatory factors secretion and chondrocyte degeneration, yet the mechanism maintaining its homeostasis remains unclear.

To Identify UFMylation system mediated by DDRGK domain-containing protein 1 (DDRGK1) as a critical regulator of STAT3 phosphorylation to prevent cartilage degeneration.

After screening differentially expressed genes and performing immunofluorescence on human cartilage, we generated cartilage-specific Ddrgk1 conditional knockout (cKo) mice and a K268R mutation at DDRGK1's key UFMylation site. Cartilage histology, RNA-seq, proteomics, phosphorylated protein microarray, western blot (WB), PCR and luciferase assays confirmed upregulated STAT3 phosphorylation. Co-IP, purified protein binding, cross-linking with mass spectrometry, and immunofluorescence explored the regulatory mechanism of STAT3 phosphorylation. A specific inhibitor was then selected for interventional treatment.

DDRGK1 expression decreased significantly in human degenerated cartilage, especially in the deep zone. Conditional knockout of Ddrgk1 in murine chondrocytes or K268 mutation exacerbated cartilage degeneration in the destabilization of medial meniscus (DMM) model. Multi-omics analysis revealed dysregulation of the STAT signaling pathway in both Ddrgk1 knockout and K268R mutant chondrocytes, with Il-6 and Stat3 as the core genes. Crosslinking MS data demonstrated direct DDRGK1-STAT3 interaction mediated by DDRGK1-K267 and STAT3-K531. Mutations in these regions disrupted their interaction. DDRGK1-K267 mediated UFMylation of STAT3 and phosphorylated STAT3, leading to impaired phosphorylation and subsequent nuclear translocation. Importantly, in human degenerated cartilage, decreased DDRGK1 expression was associated with increased phosphorylation and nuclear translocation of STAT3. This finding was recapitulated in murine models, where cKo of Ddrgk1 or K268R mutation of Ddrgk1 in chondrocytes similarly enhanced STAT3 phosphorylation and nuclear translocation. Strikingly, the specific inhibitor of STAT3 phosphorylation, SH-4-54, effectively rescued cartilage degeneration in Ddrgk1 cKo mice.

These findings unveil a novel, UFMylation-dependent role of DDRGK1 in regulating STAT3 activity and maintaining cartilage homeostasis.