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

Date Published: January 28, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Xuekun Fu, Yunyan Li, Tongling Huang, Zhiwu Yu, Kun Ma, Meng Yang, Qingli Liu, Haobo Pan, Huaiyu Wang, Junfeng Wang, Min Guan.


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.

Partial Text

Differentiation and mineralization during osteoblastogenesis of mesenchymal stem/stromal cells (MSCs) are requisites to building bone.1 Osteoblastogenesis precedes propagation of mineral onto an extracellular matrix (ECM) consisted of collagen fibrils, osteocalcin, and osteopontin (OPN) secreted by the differentiating MSCs.2 Nanocrystal nucleation of apatite subsequently forms on such mineralized ECM. By means of advanced solid‐state NMR spectroscopy and distance measurements, citrate is found strongly bound to the apatite nanocrystals in bone and accounts for about 5 wt% of bone organic fraction.3 This vast amount of citrate with its carboxylate groups, provides a huge capacity for calcium binding on apatite; thereby, stabilizing the size of nanocrystal and forming the biomineralizated network essential for bone stability, strength, and resistance to fracture.3, 4 However, the source of citrate in bone is not clearly known; in particular, how intracellular citrate metabolism and deposition is governed during apatite formation is not well defined.

Although citrate has been found universally presented in vertebrate bone and accounts for ≈80% of all citrate in the body for several decades,18 only lately has it been demonstrated as a strongly bound, integral part of the nanocomposite in bone.3 Here, we reported that citrate of bone apatite was produced by mineralized MSCs by tracing with stable isotopically labeled carbon (Figure 1). Metabolomics analysis of differentiated MSC proved that glycolytic induction of lactate production is lowered; whereas, the amount of mitochondrial TCA intermediates such as citrate derived from oxidative metabolism are increased.19 A novel model for bone apatite formation proposed that calcium and phosphorus‐containing mineral aggregates in osteoblast mitochondria, transports via vesicles to the ECM and then converts to more crystalline.9 Interestingly, we determined that accumulation of citrate precipitation was closely associated with calcium deposition and ZIP1‐mediated Zn2+ influx during osteogenesis (Figures 1, 4, and 5). These evidences imply that the transport of calcium and possibly citrate take place in mitochondrial and intracellular compartments during osteogenesis, providing a missing link in deciphering the process of normal bone apatite formation.

Our results demonstrate that zinc‐Runx2/Osterix‐ZIP1 regulation axis promotes osteoblast differentiation and apatite formation; uncover mitochondrial citrate metabolism and its relationship with zinc homeostasis during bone remodeling (Figure6). These findings highlights that mitochondrial and metabolic changes not only meet higher amounts of energy demand during osteogenic differentition of MSC, but also provide metabolic intermediates directly participating in bone apatite formation. The imbalance of zinc and citrate homeostasis upon aging lead to deleterious bone formation which may have important implications for understanding and treating pathological bone loss.

Mice: Male C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The experimental protocols were approved by the Institutional Animal Care and Use Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

The authors declare no conflict of interest.




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