Orphan nuclear receptor NR4A1 regulates both osteoblastogenesis and adipogenesis in human mesenchymal stem cells

Abstract

The nuclear receptor subfamily 4 group A member 1 (NR4A1) gene plays a crucial role in both osteoporosis and adipogenesis. The present study investigated the mechanisms by which NR4A1 influences osteoblastogenesis and adipogenesis in human bone marrow-derived mesenchymal stem cells (BMD-MSCs). NR4A1 was overexpressed or knocked down in mouse MC3T3-E1 osteoblast cells and 3T3-L1 adipocyte cells, as well as in PCS-500-012, a BMD-MSC line. The alkaline phosphatase (ALP) assay and Alizarin Red S staining were performed using MC3T3-E1 and BMD-MSCs to assess ALP activity and mineralization, while Oil Red O staining was used to assess the lipid content in 3T3-L1 cells and BMD-MSCs. Total RNA was isolated from control, NR4A1-overexpressing and NR4A1 small interfering RNA (siRNA; siNR4A1)-treated BMD-MSCs. RNA sequencing (RNA-seq) was performed to identify differentially expressed genes, followed by ingenuity pathway analysis (IPA) to determine the role of NR4A1 in osteoblastogenesis and adipogenesis. NR4A1 or Nr4a1 knockdown tended to increase ALP activity and significantly increased calcification in BMD-MSCs (P<0.005) and MC3T3-E1 cells (P<0.005), respectively. By contrast, NR4A1 or Nr4a1 overexpression significantly decreased ALP activity and calcification. NR4A1 or Nr4a1 knockdown and overexpression significantly decreased and increased adipogenesis, respectively, in BMD-MSCs (P<0.005 and <0.05, respectively) and 3T3-L1 cells (P<0.005 in both). Treatments of BMD-MSCs with an NR4A1 antagonist, 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl) methane and siNR4A1 showed similar results. RNA-seq and IPA in control, NR4A1 knockdown and NR4A1 overexpressing cells indicated that Notch signaling mediated the effects of NR4A1 in osteoblastogenesis and adipogenesis. Expression of mastermind-like transcriptional coactivator 3 was reduced in the Notch signaling pathway in cells treated with siNR4A1. In conclusion, NR4A1 suppressed osteoblastogenesis and promotes adipogenesis in human BMD-MSCs. The present study also suggested that NR4A1 plays a role in the progression of osteoporosis and adipogenesis by modulating the Notch signaling cascade.