ITRAQ-based quantitative proteomic analysis of MG63 in response to HIF-1α inducers.

Non-healing fractures constitute a serious clinical problem. HIF-1α is a crucial regulator in response to hypoxia and is proven to be pivotal in bone growth; however, the mechanism still needs further research. In this study, iTRAQ was used to study the effects of two HIF-1α inducers on the expression of proteins in MG63 cells. A total of 841 proteins were significantly changed after treatment with HIF-1α inducers. Among these, 12 proteins were functionally involved in the HIF-1 and VEGF signaling pathways. We then studied the protein and gene expression of the twelve proteins by western blot and RT-PCR, respectively. The results confirmed that VEGF, TFRC, ERK1/2, iNOS, GLUT1, ALDOA, ENO1 and IP3R1 were markedly upregulated, while NF-κB, RCN1, PLCγ1 and CaMKII were significantly downregulated upon treatment with HIF-1α inducers. Meanwhile, the intracellular levels of Ca2+, NO and ROS were closely related and significantly changed. Up-regulation of HIF can maintain high levels of Ca2+ and NO while reducing ROS and protect cells from apoptosis induced by low serum. This study presents a new way to study the regulation of HIF on bone growth by investigating the Ca2+, NO and ROS levels. SIGNIFCANCE: We found that the regulation of Ca2+ and NO proteins are tightly associated with HIF pathway using iTRAQ method. Furthermore, the concentration of Ca2+, NO and ROS are closely related in low serum cultured cells. Up-regulation of HIF pathway can maintain high levels of Ca2+ and NO while reducing ROS damage.