High-Throughput Preosteoblastic Spheroids Elevate Fibroblast Growth Factor 23 via Parathyroid Hormone Signaling Pathway

Abstract

Fibroblast growth factor 23 (FGF23) plays a crucial role in managing renal phosphate and the synthesis of 1,25(OH)2-vitamin D3, which is essential for bone homeostasis. Developing robust in vitro systems to study FGF23-regulating mechanisms is crucial for advancing our knowledge and identifying potential therapeutic targets. The traditional in vitro 2D culture system results in relatively low expression of FGF23, complicating further exploration of its regulatory mechanisms and potential therapeutic targets. Herein, we reported a high-throughput approach to generate preosteoblastic cell spheroids with enhanced FGF23 production. For this purpose, murine preosteoblast cell line (MC3T3-E1) was cultured in our previously reported nonadherent microwells (200 µm in diameter, 148 µm in depth, and 100 µm space in between) and self-assembled into spheroids with a diameter of 92.3 ± 15.0 µm after 24 h. Compared with monolayer culture, the MC3T3-E1 spheroids showed a significant upregulation of FGF23 in both gene and protein levels after 24 h of serum-free induction. RNA sequencing and western blotting analysis further suggested that the enhanced FGF23 production in MC3T3-E1 spheroids was attributed to the activation of the parathyroid hormone (PTH)/PTH1R signaling pathway. Impressively, inhibition of PTH signaling through small molecular inhibitors or short hairpin RNA targeting PTH1R effectively reduced FGF23 production. In summary, the current study revealed the efficacy of the high-throughput formation of preosteoblast cell spheroid in stimulating FGF23 expression for mechanistic studies. Importantly, our findings highlight the potential of the current 3D spheroid system for target identification and drug discovery.

Impact Statement

This research introduces a high-throughput method for generating preosteoblastic spheroids that significantly enhance fibroblast growth factor 23 (FGF23) production via the parathyroid hormone signaling pathway. The findings offer a robust in vitro model for studying FGF23 regulation, addressing the limitations of traditional 2D cultures. This advancement not only improves our understanding of bone and mineral metabolism but also holds potential for identifying novel therapeutic targets for conditions with elevated FGF23 levels, such as chronic kidney disease and metabolic bone disorders. The scalable and efficient spheroid formation method could revolutionize the approach to drug discovery and target validation for managing FGF23 production.