Examining the Role of HREs on the Regulation of Opioid Receptor Gene Expression in Neuronal Cells undergoing Hypoxic Mimic Condition

Hypoxia is a condition of inadequate oxygen supply, which can induce cell death. Using human neuronal cells treated with a hypoxia memetic compound, desferoxamine (DFO), a hypoxic cell model system was created. Our lab reported previously that treatment with DFO resulted in the decrease of cell viability. However, there were still surviving neurons. The surviving cells did not exhibit significant morphological changes, as compared to the control cells, under confocal microscopy analysis using annexin-V-FLUOS and propidium iodide staining, indicating that they were not at apoptotic or necrotic stages. These surviving neurons, therefore, developed adaptive responses under hypoxic challenge. Several changes were observed, including an increase of cellular glutathione level and the increase of hypoxia inducible factor 1-α (HIF-1α) mRNA level, a known marker of hypoxia, in surviving cells when compared to those of control cells. The increase of human kappa opioid receptor (hKOR) expression was also observed in surviving neurons when compared to the control cells. Clinically, opioids are used to modulate pain sensation, which can result from events such as stroke, trauma and cardiac arrest that lead to hypoxic conditions. There are 4 potential HIF response elements found in the 5’ upstream region of this gene. Two of the four elements have shown a significant increase of reporter activity in cells undergoing DFO-induced hypoxic conditions. To determine if these two elements displayed an interactive effect under hypoxic challenge, they were simultaneously cloned into a reporter plasmid containing the luciferase gene, and resulting plasmids were tested using NMB neuronal cells. Results showed a significant increase of promoter activity under hypoxic challenge. Mutation analysis further confirmed the important role of these elements. Taken together, this study showed the functional roles of two HIF response elements mediating the kappa opioid receptor expression under hypoxic condition