MFN2-Mediated MERCs Promote Odontoblast Differentiation and Dentinogenesis

Odontoblasts are a tooth-specific cell type responsible for dentin formation. During odontoblast differentiation, dramatic organelle alterations occur with the increase of both mitochondria and endoplasmic reticulum (ER). In multiple cell types, mitochondria–ER contact sites (MERCs) are formed to regulate cellular biological processes via mediating essential interorganelle communications. However, whether MERCs play a role in odontoblast differentiation remains enigmatic. Here, we found a progressive increase of MERCs during the in vitro odontoblastic differentiation using live-cell imaging, transmission electron microscopy (TEM), and in situ proximity ligation assay (PLA). Meanwhile, in situ PLA verified the in vivo elevation of MERCs in the odontoblasts of mouse teeth. Remarkably, the disruption of MERCs via overexpression of FATE1, a protein that increases the distance between mitochondria and ER, suppressed the expression of key odontoblast markers, alkaline phosphatase (ALP) activity, and mineralized nodule formation in the mouse dental papilla cells (mDPCs) after differentiation induction, suggesting a positive role of MERCs for odontoblastic differentiation. Among all MERC-associated genes, Mfn2 expression is significantly increased in the RNA sequencing data of odontoblast-like cells versus undifferentiated mDPCs. Further experiments showed that knockdown of Mfn2 resulted in diminished MERC formation as well as impaired odontoblastic differentiation, as evidenced by the downregulated expression of odontoblast markers, compromised ALP activity, and mineralized nodule formation. The impaired odontoblastic differentiation upon Mfn2 knockdown was rescued by the overexpression of Linker, an enhancer of MERC formation, suggesting that MFN2 enhances MERC formation to promote odontoblastic differentiation. Consistently, in vivo knockdown of Mfn2 also impaired MERC formation as well as odontoblast differentiation and dentinogenesis, which were restored by in vivo Linker overexpression. Taken together, these findings indicate that MERCs are dynamically increased and essential for odontoblast differentiation and dentinogenesis with MFN2 as an essential mediator, which provides an important mechanism of organelle interaction orchestrating odontoblast differentiation.