Effects of desferoxamine-induced hypoxia on neuronal human mu-opioid receptor gene expression.
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.
Cook
Ryan J
RJ
Department of Biological Sciences, Seton Hall University, South Orange, NJ 07079, United States.
Karch
Christopher
C
Nahar
Pranjal
P
Rivera
Aida
A
Ko
Jane L
JL
D015687:Cell Hypoxia / Q000235:genetics; D045744:Cell Line, Tumor; D003676:Deferoxamine / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D000860:Hypoxia / Q000139:chemically induced / Q000235:genetics*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D020871:RNA Stability; D017450:Receptors, Opioid, mu / Q000235:genetics*
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017450:Receptors, Opioid, mu; D008156:Luciferases; D003676:Deferoxamine