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

The nuclear receptor subfamily 4 group A member 1 (<i>NR4A1</i>) 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). <i>NR4A1</i> 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, <i>NR4A1</i>‑overexpressing and <i>NR4A1</i> small interfering RNA (siRNA; <i>siNR4A1</i>)‑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 <i>NR4A1</i> in osteoblastogenesis and adipogenesis. <i>NR4A1</i> or <i>Nr4a1</i> 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, <i>NR4A1</i> or <i>Nr4a1</i> overexpression significantly decreased ALP activity and calcification. <i>NR4A1</i> or <i>Nr4a1</i> 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 <i>siNR4A1</i> showed similar results. RNA‑seq and IPA in control, <i>NR4A1</i> knockdown and <i>NR4A1</i> overexpressing cells indicated that Notch signaling mediated the effects of <i>NR4A1</i> in osteoblastogenesis and adipogenesis. Expression of mastermind‑like transcriptional coactivator 3 was reduced in the Notch signaling pathway in cells treated with <i>siNR4A1</i>. In conclusion, <i>NR4A1</i> suppressed osteoblastogenesis and promotes adipogenesis in human BMD‑MSCs. The present study also suggested that <i>NR4A1</i> plays a role in the progression of osteoporosis and adipogenesis by modulating the Notch signaling cascade.