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

Abstract

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.

Citrate chemistry and biology for biomaterials design

“…All the above studies demonstrate that citrate is a strongly bound and integral part of native bone, serving as a key mineralization regulator. Intriguingly, the citrate involved in bone mineralization has been found to be derived from intracellular citrate metabolism of differentiating osteoblasts, as uncovered by a recent study [121]. Specifically, in response to osteogenic stimulation, hMSCs undergo metabolic reprogramming with a metabolic shift from glycolysis to oxidative respiration [122].…”

“…Specifically, in response to osteogenic stimulation, hMSCs undergo metabolic reprogramming with a metabolic shift from glycolysis to oxidative respiration [122]. Meanwhile, gene expression of citrate synthase (CS) and its activity [121, 123] elevate along with increased uptake of zinc, which inhibits aconitase-mediated citrate oxidation [121, 124–127]. Collectively, it leads to citrate accumulation in mitochondria, which subsequently exported via CIC to extracellular spaces incorporated in newly formed bone.…”

Zinc Homeostasis in Bone: Zinc Transporters and Bone Diseases

“…induction of osteogenesis of MSCs [39][40][41] knockout no obvious bone phenotype [42][43][44] ZIP2 knockout no obvious bone phenotype [43][44][45] ZIP3 knockout no obvious bone phenotype [42][43][44] ZIP8 cartilage destruction and osteoarthritis [46][47][48][49] ZIP13 mutation spondylocheiro dysplastic form of Ehlers-Danlos syndrome [50] knockout connective tissue dysplasia [51] ZIP14 knockout growth retardation [52,53] mutation hyperostosis cranialis interna [54] ZnT5 knockout poor growth, osteopenia and heart failure [55] ZnT7 impaired osteogenesis of MSCs [56] protection of osteoblasts from apoptosis [57]…”

“…Additionally, ZIP1 is located in the cytoplasmic membrane and mediates zinc influx during mesenchymal stem cell (MSC) differentiation into osteoblasts [40]. ZIP1 expression was induced upon osteogenic differentiation of multipotent human MSCs and osteoblast progenitor MC3T3-E1 cells in vitro, accompanied by an increase in intracellular zinc uptake [39][40][41]. Moreover, ZIP1-mediated zinc influx gave rise to higher expression of the osteogenic master regulators Runx2 and Osterix.…”

“…Enhancement of Runx2 and Osterix expression further modulated transcriptional expression of ZIP1 by directly binding to the response elements in the promoter. These processes resulted in a series of intertwined feed-forward loops that induced zinc influx and osteogenic differentiation and promoted bone apatite formation [41,61,62]. On the other hand, ZIP1 is also expressed in osteoclasts, and overexpression of ZIP1 inhibited mature osteoclast activity by exerting a negative impact on the nuclear factor kappa B (NF-κB) pathway, which is essential for osteoclast differentiation and activity [63].…”