SIAH1-Mediated Ubiquitination and Degradation of TRIB3 Modulate Osteogenic and Adipogenic Differentiation.

Background: The imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is a key factor in osteoporosis (OP). TRIB3 acts as a molecular switch regulating this process. While E3 ubiquitin ligases are known to influence TRIB3 protein stability, the specific role of E3 ligase-mediated TRIB3 ubiquitination in directing BMSCs' fate remains unclear. This study systematically investigates this mechanism. Methods: We first predicted potential E3 ubiquitin ligases for TRIB3 using the UbiBrowser database. Differentially expressed E3 ligases were then screened from the GEO datasets. Molecular docking with the TRIB3 protein identified the candidate with the highest binding affinity for a mechanistic study. Serum samples from osteoporotic patients and healthy controls were analyzed to verify clinical correlation. For in vivo validation, we performed in vivo rescue experiments in naturally aged osteoporotic mice using femoral injection of SIAH1-knockdown lentivirus. Immunohistochemistry, immunofluorescence, and western blot were used to assess the expression of TRIB3 and SIAH1. In vitro, we overexpressed or knocked down SIAH1 to examine its effects on TRIB3 ubiquitination, protein stability, and BMSCs differentiation. TRIB3 rescue assays were performed to confirm the dependency. Results: UbiBrowser analysis identified eight E3 ubiquitin ligases targeting TRIB3. Subsequent screening selected SIAH1 for mechanistic investigation. In aged osteoporotic mice and osteoporotic patients, SIAH1 expression was significantly increased, while TRIB3 was decreased. In vivo rescue experiments showed that local knockdown of SIAH1 restored TRIB3 expression, improved bone mineral density (BMD), trabecular microarchitecture, and rebalanced osteogenic and adipogenic differentiation. In vitro, upregulating SIAH1 promoted TRIB3 ubiquitination and proteasomal degradation, reducing TRIB3 protein levels, downregulating osteogenic markers, and upregulating adipogenic markers. Knocking down SIAH1 yielded the opposite effects. TRIB3 overexpression or knockdown fully reversed the effects of SIAH1 on BMSC differentiation. Conclusion: SIAH1 promotes the ubiquitination and proteasomal degradation of TRIB3, thereby disrupting the osteogenic-adipogenic balance of BMSCs and contributing to age-related bone loss. Local inhibition of SIAH1 stabilizes TRIB3 and ameliorates OP. These findings provide a scientific basis and novel therapeutic target for regulating BMSCs differentiation and treating age-related OP via the SIAH1-TRIB3 axis.