MLK3 silence suppressed osteogenic differentiation and delayed bone formation via influencing the bone metabolism and disturbing MAPK signaling

BACKGROUND: Mixed lineage kinase 3 (MLK3) is a member of a serine/threonine MAP3K family, and it has been demonstrated to play critical roles in various biological activities and disease progression. Previous studies showed that impaired skeletal mineralization and spontaneous tooth fracture in the MLK3-deficient mice, suggesting MLK3 actively participated in the bone formation. However, the detailed function and underlying mechanisms remain obscure. METHODS: The MLK3 knockout (KO) mouse was applied in the present study, and multi-omics were performed to compare the metabolites and gene expression between wild type (WT) and KO mice. The bone fracture model was successfully established, and the healing process was evaluated by X-ray, micro-CT examination, histomorphometry and immunohistochemistry (IHC) staining. On the other hand, the effects of MLK3 on osteogenic differentiation were assessed by alkaline phosphatase (ALP) activity, Alizarin red S (ARS) staining and qRT-PCR examination. Finally, the downstream signaling pathways were screened out by RNA-sequencing (RNA-seq) and then validated by Western blotting. RESULTS: In the present study, imbalanced bone metabolism was observed in these MLK3 KO mice, suggesting MLK3 may participate in bone development. Moreover, MLK3 −/− mice displayed abnormal bone tissues, impaired bone quality, and delayed fracture healing. Further investigation showed that the inhibition of MLK3 attenuated osteoblast differentiation in vitro. According to the RNA-seq data, MAPK signaling was screened out to be a downstream pathway, and its subfamily members extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal protein kinase (JNK) were subjected to Western blotting examination. The results revealed that although no differences in their expression were observed between MSCs derived from WT and KO mice, their phosphorylated protein levels were all suppressed in MLK3 −/− MSCs. CONCLUSION: In conclusion, our results demonstrated that loss of MLK3 suppressed osteoblast differentiation and delayed bone formation via influencing metabolism and disturbing MAPK signaling. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The findings based on the current study demonstrated that MLK3 promoted osteogenesis, stimulated new bone formation and facilitated fracture healing, suggesting that MLK3 may serve as a potential therapeutic target for bone regeneration. MLK3 activator therefore may be developed as a therapeutic strategy for bone fracture.