Expression of dual specificity phosphatase-1 in hypoxic RAW264.7 cells and its role in inflammatory immune response regulation

Abstract

Objective To investigate the expression of dual specificity phosphatase-1 (DUSP1) in hypoxic RAW264.7 macrophages and its regulatory role in hypoxia-induced RAW264.7 macrophage inflammatory immune response. Methods RAW264.7 macrophages were cultured under a hypoxic condition (1%O2) for 12, 24 and 36 h, with the cells maintained in routine culture conditions (21%O2) as control. RNA interference technique was used to inhibit Dusp1 expression in RAW264.7 cells, which were exposed to hypoxia for 24 h following the transfection. The cells and the culture supernatant were collected after the treatments, and the mRNA expression levels of Dusp1, Il1b, Il10 and Tnfa were detected using qRT-PCR; and the protein expression of DUSP1 was detected using Western blotting, and the levels of interleukin-1β (IL-1β), IL-10 and tumor necrosis factor-α (TNF-α) in the supernatant were determined with ELISA. Results Compared with the control cells cultured in normoxic condition, RAW264.7 cells with hypoxic exposures exhibited significantly increased the mRNA and protein levels of DUSP1 (P < 0.05), whose protein expression reached the peak level at 24 h of hypoxia. The mRNA levels of Il1b and Il 10increased significantly after hypoxic exposure for 24 and 36 h (P < 0.05), and the expression of Tnfa mRNA reduced significantly after a 12-h exposure (P < 0.05). Hypoxic exposure caused a significant increase in the secretion level of IL-1β by the macrophages (P < 0.05). Transfection with si-Dusp1 significantly increased the mRNA expression of Il1b and the secretion levels of IL-1β and IL-10 and significantly lowered the mRNA expression and secretion level of TNF-α in hypoxic RAW264.7 cells (all P < 0.05). Conclusion Hypoxia can up-regulate DUSP1 expression in RAW264.7 macrophages. DUSP1 participates in the regulation of hypoxia-induced RAW264.7 macrophage inflammatory immune response by modulating the expression and the secretion of IL-1β, IL-10 and TNF-α.

Keywords

• dual specificity phosphatase-1
• hypoxia
• raw264.7 cells
• inflammatory immune response