Effects of desferoxamine-induced hypoxia on neuronal human mu-opioid receptor gene expression

Abstract

The effect of desferoxamine (DFO)-induced hypoxia on neuronal human mu-opioid receptor (hMOR) gene expression was investigated using NMB cells. DFO decreased cell viability and increased cellular glutathione levels in a dose- and time-dependent manner. Confocal analysis using annexin-V-fluorescein and propidium iodide staining revealed that surviving/attached cells under DFO challenge were morphologically similar to control (vehicle-treated) cells. RT-PCR analysis demonstrated that the hypoxia inducible factor-1alpha (HIF-1alpha) mRNA level was augmented in these surviving neurons. DFO treatment for 8h or longer down-regulated the hMOR message, but not that of the delta-opioid receptor. Functional analysis using luciferase reporter assay showed that the hMOR 5'-regulatory region, from -357bp to translational initiation site (+1), contains the active promoter with an inhibitory region located in the -422 to -357bp region. DFO decreased hMOR promoter activity as compared to control. Mutation analysis suggested the existence of both dsDNA and ssDNA elements, located in a CT-rich region of hMOR, mediating the DFO-response. RT-PCR further revealed that DFO exhibited no effect on hMOR mRNA stability. In conclusion, DFO-induced hypoxia specifically affects neuronal hMOR gene expression at the transcriptional, not post-transcriptional, level.