Runx2/Osterix and Zinc Uptake Synergize to Orchestrate Osteogenic Differentiation and Citrate Containing Bone Apatite Formation

Citrate is essential to biomineralization of the bone especially as an integral part of apatite nanocomposite. Citrate precipitate of apatite is hypothesized to be derived from mesenchymal stem/stromal cells (MSCs) upon differentiation into mature osteoblasts. Based on ¹³C-labeled signals identified by solid-state multinuclear magnetic resonance analysis, boosted mitochondrial activity and carbon-source replenishment of tricarboxylic acid cycle intermediates coordinate to feed forward mitochondrial anabolism and deposition of citrate. Moreover, zinc (Zn²⁺) is identified playing dual functions: (i) Zn²⁺ influx is influenced by ZIP1 which is regulated by Runx2 and Osterix to form a zinc-Runx2/Osterix-ZIP1 regulation axis promoting osteogenic differentiation; (ii) Zn²⁺ enhances citrate accumulation and deposition in bone apatite. Furthermore, age-related bone loss is associated with Zn²⁺ and citrate homeostasis; whereas, restoration of Zn²⁺ uptake alleviates age-associated declining osteogenic capacity and amount of citrate deposition. Together, these results indicate that citrate is not only a key metabolic intermediate meeting the emerging energy demand of differentiating MSCs but also participates in extracellular matrix mineralization, providing mechanistic insight into Zn²⁺ homeostasis and bone formation.