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Triamcinolone acetonide destabilizes VEGF mRNA in Müller cells under continuous cobalt stimulation
Jonathan E Sears1, George Hoppe
1Cole Eye Institute, Cleveland Clinic Foundation, OH 44195, USA. searsj@ccf.org
Purpose:
To identify the molecular mechanism of steroid-induced downregulation of vascular endothelial growth factor (VEGF) synthesis in Müller cells.
Methods:
Confluent cultures of human Müller cells (MIO-M1) were treated with 100 microM CoCl(2), 1 microg/mL triamcinolone acetonide (TA), or both. VEGF secretion was measured with respect to time by ELISA. VEGF mRNA quantity and stability were analyzed by reverse transcriptase-polymerase chain reaction. The activity of hypoxia-inducible factor (HIF)-1 was measured by the relative binding of HIF-1 protein to the hypoxia response element (HRE), by gel shift and ELISA. The HIF-1alpha protein level was determined with Western blot.
Results:
TA decreased VEGF secretion by at least 50% in the presence of continuous cobalt stimulus. VEGF mRNA decreased 50- to 100-fold 6 hours after treatment with TA and cobalt compared with cobalt alone. VEGF mRNA stability was decreased in cobalt-stimulated, TA-treated cells compared with cobalt alone in cells synchronized by exposure to actinomycin D. HIF-1alpha protein level was sustained for the entire 24-hour treatment period and partitioned into nuclear, not cytosolic, fractions. HIF-1 activity was decreased by 20% to 30% in the presence of TA and cobalt compared with cobalt alone.
Conclusions:
TA may decrease VEGF synthesis by nongenomic destabilization of VEGF mRNA in cobalt-stimulated Müller cells. There was little effect on the total HIF-1alpha protein level, HIF-1 partitioning, and HIF-1 activity.
Insights
Triamcinolone acetonide (TA) reduces vascular endothelial growth factor (VEGF) synthesis in Müller cells by destabilizing VEGF mRNA. This steroid effect occurs independently of major changes in hypoxia-inducible factor-1 (HIF-1) activity or protein levels.
Area of Science:
- Ophthalmology
- Molecular Biology
- Cell Biology
Background:
- Vascular Endothelial Growth Factor (VEGF) is crucial for retinal neovascularization.
- Steroids like triamcinolone acetonide (TA) are used to treat ocular conditions involving VEGF.
- The precise molecular mechanism of steroid-induced VEGF downregulation in Müller cells remains unclear.
Purpose of the Study:
- To elucidate the molecular mechanism by which steroids, specifically TA, downregulate VEGF synthesis in human Müller cells.
- To investigate the role of hypoxia-inducible factor-1 (HIF-1) in this process.
Main Methods:
- Human Müller cells (MIO-M1) were treated with cobalt chloride (CoCl2) to induce hypoxia and TA.
- VEGF secretion was quantified using ELISA.
- VEGF mRNA levels and stability were assessed via RT-PCR.
- HIF-1 activity and HIF-1alpha protein levels were analyzed using gel shift assays, ELISA, and Western blotting.
Main Results:
- TA significantly reduced VEGF secretion and mRNA levels in cobalt-stimulated Müller cells.
- VEGF mRNA stability was decreased by TA treatment, suggesting post-transcriptional regulation.
- HIF-1alpha protein levels remained stable, and HIF-1 activity showed only a minor decrease with TA treatment.
Conclusions:
- TA likely decreases VEGF synthesis through non-genomic destabilization of VEGF mRNA in Müller cells.
- The mechanism does not appear to involve significant alterations in HIF-1alpha protein levels, nuclear translocation, or overall activity.
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