DDIT3 switches osteogenic potential of BMP9 to lipogenic by attenuating Wnt/β-catenin signaling via up-regulating DKK1 in mesenchymal stem cells

Bone morphogenetic protein 9 (BMP9) functions as a potent inducer of osteogenic differentiation in mesenchymal stem cells (MSCs), holding promise for bone tissue engineering. However, BMP9 also concurrently triggers lipogenic differentiation in MSCs, potentially compromising its osteogenic potential. In this study, we explored the role of DNA damage inducible transcript 3 (DDIT3) in regulating the balance between BMP9-induced osteogenic and lipogenic differentiation in MSCs. Utilizing techniques such as PCR, Western blot, histochemical staining, and <i>in vivo</i> experiments, we analyzed the osteogenic and lipogenic markers induced by BMP9 and delved into the underlying molecular mechanism. We found a significant upregulation of DDIT3 in C3H10T1/2 cells treated with BMP9. This upregulation led to a reduction in BMP9-induced osteogenic markers but an enhancement in lipogenic markers. Conversely, knocking down DDIT3 produced the opposite effects. Furthermore, BMP9-induced bone formation was decreased in the presence of DDIT3, but adipocyte formation was increased. Further investigations demonstrated that BMP9 increased the phosphorylation level of GSK-3β and promoted nuclear translocation of β-catenin, both of which were suppressed by DDIT3. Moreover, DDIT3 decreased the total β-catenin protein level while BMP9 increased the DKK1 protein level, which was further enhanced by DDIT3. Notably, knocking down DKK1 partially reversed the effect of DDIT3 on reducing BMP9-induced osteogenic markers and increasing lipogenic markers. Our findings indicated that DDIT3 enhances lipogenic differentiation by diminishing BMP9's osteogenic potential, possibly through inhibiting Wnt/β-catenin signaling via DKK1 upregulation in MSCs.