Abstract
Background: The differentiation process of human mesenchymal stem cells (hMSCs) is regulated by a variety of chemical, physical, and biological factors. These factors activate distinct signaling pathways and transcriptional networks, thereby regulating the lineage-specific differentiation of hMSCs. Objective: This study aims to investigate the role of Immediate Early Response 3 (IER3) in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and explore the underlying regulatory mechanisms by which IER3 influences osteogenesis. Methods: The expression levels of IER3 and osteogenesis-related genes were quantified when hMSCs were subjected to in vitro osteogenic induction. Then, stable IER3-knockdown hMSCs were generated using IER3-targeted shRNA lentiviral vectors, and the impact of IER3 on osteogenic differentiation was evaluated through both in vitro cell induction and hMSCs subcutaneous implantation model of nude mice. Moreover, RNA-seq and functional inhibition assays were performed to elucidate the signaling pathway through which IER3 regulates the osteogenic differentiation of hMSCs. Results: IER3 expression was significantly downregulated during osteogenic differentiation. Knockdown of IER3 markedly upregulated the expression of ALP and RUNX2, enhancing the osteogenic differentiation capacity of hMSCs, both in vitro and in vivo. Mechanistic studies revealed that IER3 knockdown significantly increased phosphorylated ERK1/2 levels, activating the MAPK/ERK signaling pathway. Furthermore, inhibition of the MAPK/ERK signaling pathway reversed the enhanced osteogenic differentiation observed following IER3 knockdown. Conclusions: Knockdown of IER3 promotes osteogenic differentiation of hMSCs through regulation of the MAPK/ERK signaling pathway, indicating IER3 represents a potential therapeutic target for the treatment of osteoporosis and bone defect-related diseases.