HIF-1A and C/EBPs transcriptionally regulate adipogenic differentiation of bone marrow-derived MSCs in hypoxia

Chen Jiang, Jun Sun, Yafei Dai, Pengfei Cao, Liyang Zhang, Shuping Peng, Yanhong Zhou, Guiyuan Li, Jingqun Tang, Juanjuan Xiang

Bone marrow-derived mesenchymal stem cells (BMSCs, also known as bone marrow-derived mesenchymal stromal cells) are known to be a component of the tumor microenvironment. BMSCs are multipotent stromal cells that can differentiate into a variety of cell types, including osteocytes, chondrocytes, adipocytes, epithelial cells and endothelial cells. Stem cells found in niches or transplanted into injured tissues constantly encounter hypoxic stress. Areas with very low to no oxygen pressure exist in solid tumors. The differentiation capacity of BMSCs under hypoxic conditions remains controversial. In this study, a hypoxic workstation, set at an oxygen concentration of 0.2% was used to mimic the hypoxic microenvironment of cancer in vivo. Oil red O staining and alkaline phosphatase staining were used to examine the adipogenic or osteogenic differentiation, respectively, of BMSCs. Real-time PCR was performed to explore the expression of adipocyte- or osteocyte-specific genes. An RT2 Profiler(TM) PCR Array was used to screen a panel of 84 genes associated with human adipogenesis in BMSCs under normal and hypoxic conditions. A dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were applied to analyze promoter activity to evaluate the possible regulatory mechanism of adipocyte-specific gene expression. We found that this extreme hypoxia impaired osteogenic differentiation as indicated by the attenuation of alkaline phosphatase (ALP) activity and the reduced expression of osteogenic markers osteocalcin and osteopontin. Moreover, extreme hypoxia enhanced adipogenic differentiation, as indicated by the accumulation of lipid droplets and the expression of the adipocyte-specific genes leptin, LPL, CFD, PGAR and HIG2. In the extreme hypoxic conditions (0.2% oxygen), the overexpression of CCAAT enhancer-binding proteins (C/EBPs), especially C/EBPδ, and HIF-1A upregulated the promoter activities of adipocyte-specific genes such as leptin, CFD, HIG2, LPL, PGAR. In the present study, peroxisome proliferator-activated receptor-gamma (PPARγ) exerted a negative effect on the differentiation of BMSCs into adipocytes. In view of these findings, extreme hypoxia induced the adipogenic differentiation of BMSCs through HIF-1A and C/EBPs. These findings might provide clues regarding the roles of BMSCs in the cancer microenvironment.