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.

Fig. 1

Effect of DFO on NMB cells A-B, Cells were treated without (control) or with different concentrations of DFO for 24 hrs (A), or 200μM DFO for 2, 4 or 24 hrs (B). Total number of attached cells was determined. Cell viability (open circles in A; gray bars in B) is presented as a percentage of total cell number from DFO-treated group divided by the number from control (as 100%). Cellular glutathione level (closed squares in A; black bars in B) is present as the percentage of the average amount of glutathione per cell from DFO-treated group divided by control (as 100%). Data is present as mean± S.E. “*” indicates p< 0.01 (student paired t-test). C and D, Surviving/attached cells under DFO, no treatment (Control), or H₂O₂ treatment (positive control) were stained using annexin-V-FLUOS (as FITC in green color) and propidium iodide (as PI in red color). Cells were imaged using confocal microscope under 10x in C or 40x magnification of object lens in D. Merged images are overlapped images of transmitted light (TL) with FITC and PI images, or an overlapped image (panel f in D) of FITC and PI images. An arrow indicates the cell staining by both annexin-V and PI.

Fig. 2

DFO alters endogenous HIF-1α and hMOR message levels RNA was extracted from cells treated without (control, C) or with DFO (A-C) for 24 hrs, or with DFO for 0, 2, 4, 8, 12 or 24 hrs (D-E). RT-PCR was performed using human HIF-1α (A, D), MOR (B, E) or DOR (C)-specific primers. Human β-actin specific primers were included in every PCR reaction (added at the cycle 19) as an internal control for normalization purpose. The normalized message from control, or from cells at time zero, was arbitrarily defined as 100%. Quantitative analysis of message levels are presented as mean ± S.E. “*” indicates p< 0.01. (student paired t-test).

Fig. 3

Identification of hMOR gene promoter and DFO-effect on promoter activity Various lengths of hMOR 5′-regulatory regions were cloned into the promoterless luciferase vector, pGL3-basic (B). Numbers on the left and right of each plasmid refer to the nucleotide number at the 5′- and 3′-ends, of each inserted fragment with its orientation indicated by the arrow. Reverse orientation is shown by an arrow pointing toward left. A deletion is indicated by a dashed line. Translation initiation site is designated as +1, and the transcription initiation site (TIS) is −216 bp. Transient transfection and luciferase assays were performed. A and C, Promoter activity of each construct was expressed as n-fold activation of pGL3-basic plasmid activity. Histograms represent mean values of fold activation. Error bars indicate S.E. “*” indicates p< 0.01 (student paired t-test). B, Sequence comparison of hMOR 5′-regulatory and mMOR proximal promoter regions. Open box represents the CT-rich region, with C/T transition underlined. D-E, “X” indicates the mutated ss or ds element. DNA sequences of wild type (h325/285) and mutant (h325/285-ds with GGT mutation; h325/285-ss with the GC box substitution underlined) are shown. Cells, transfected with plasmid, were treated without (control) or with DFO for 24hrs. Luciferase activity from control was defined as 100%. Data are presented as mean ± S.E. “*” indicates p< 0.01.

Fig. 4

Effect of DFO on hMOR mRNA stability RT-PCR was performed using hMOR-specific primers with RNA from cells treated with Actinomycin D (ActD) or DFO plus ActD for 0, 2, 4, 8, 12 or 24 hrs. The β-actin was used as an internal control. PCR products were resolved using electrophoresis. Sizes of DNA markers are indicated. Quantitative analysis of hMOR message levels are presented as mean ± S.E., with normalized hMOR message at time zero as 100%. “*” indicates p< 0.01 (student paired t-test).