Osteogenesis is well regulated by various cytokines and signaling pathways. Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGF-β) super-family, and several BMP members possess osteogenic potential. To date, BMP9 is the most effective osteogenic BMP and may be a promising alternative for bone tissue engineering. However, the details of how BMP9 induces osteogenic differentiation in mesenchymal stem cells (MSCs) remain unclear.

Metabolic reprogramming, such as re-balancing of glycolysis and oxidative phosphorylation, is important for cell proliferation and differentiation (Ryall, 2013). Pyruvate dehydrogenase complex converts pyruvate to acetyl-CoA, which is oxidized in mitochondria to produce energy or other intermediate metabolites. Pyruvate dehydrogenase kinase 4 (PDK4) phosphorylates pyruvate dehydrogenase and decreases its activity. PDK4 plays a critical role in promoting calcification of vascular smooth muscle cells (VSMCs), which may be mediated via enhanced SMAD1/5/9 phosphorylation (Leem and Lee, 2016). VSMC calcification shares certain physiological processes with the differentiation of MSCs to osteoblasts, such as increased BMP/SMAD signaling activity (Lee et al., 2015). Our previous studies demonstrated that cyclooxygenase-2 (COX-2) was involved in high phosphate-induced calcification of VSMCs as well as BMP9-induced osteogenic differentiation of MSCs (He et al., 2018, Wang et al., 2013). Since BMP9 is involved in regulating glucose metabolism, fatty acid, and nerve metabolism (Chen et al., 2003, López-Coviella et al., 2000, Wang et al., 2020), PDK4 may play an important role in BMP9-induced osteogenic differentiation of MSCs.

Wnt/β-catenin signaling plays an important role in regulating bone development and metabolism. BMP9-induced osteogenic differentiation was enhanced by activating Wnt/β-catenin signaling, and significantly reduced by inhibiting this pathway (Tang et al., 2009, Zhang et al., 2015). Retinoic acid causes enhanced BMP9-induced osteogenic differentiation through increased activity of Wnt/β-catenin signaling in MSCs (Wang et al., 2019). Furthermore, retinoic acid signaling regulates the PDK4 expression. It remains unknown whether the effect of BMP9 on activating Wnt/β-catenin signaling is associated with PDK4 in MSCs.

The C3H10T1/2 cell line, a type of MSC, was established from C3H mouse embryos. This cell line has been commonly used for directed differentiation studies, particularly those related to osteogenic, chondrogenic, and adipogenic differentiation (Tang et al., 2004). Therefore, we used the C3H10T1/2 cell line to investigate the possible role of PDK4 in BMP9-triggered osteoblastic differentiation. Moreover, we explored the effect of PDK4 on BMP9-induced Wnt/β-catenin signaling activation as well as the underlying possible mechanism.