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Alina Genis, Sandeep Kumar, John Danias; Decreased PLAT expression and PLAT Promoter Activity after Steroid Treatment. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5657.
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Steroids downregulate PLAT, the gene encoding for tissue Plasminogen Activator (tPA,) although the exact mechanism through which this occurs is unknown. We investigated steroid-induced changes in PLAT expression to better characterize this interaction.
Human TM cells were generated from donor rings left over after penetrating keratoplasty. Cells were confirmed to express myocilin, SMA and aB-crystalin. Cells up to passage 4 were treated with Prednisolone Acetate (PA) (80 ng/ml) for periods up to 24 hours. PLAT expression was determined using qPCR. A plasmid containing the firefly luciferase gene under the control of the human PLAT promoter regulatory region (~800 bp long upstream of ATG) was used to transfect HTM cells, via electroporation, after which cells were subjected to PA treatment as above. Cells were also co-transfected with another plasmid containing the gene for secreted Renilla luciferase under the control of a CMV promoter. Luciferase activity was measured 24 hours later and PLAT promoter expression was normalized to the Renilla luciferase expression to account for transfection efficiency.
PLAT expression was significantly decreased as early as 1 hour after steroid treatment in cells from one of the donors (p<0.05.) Steroid treated cells from the same donor also showed a significant decrease in the PLAT driven luciferase expression (P<0.05.)
Steroids decrease PLAT expression in HTM cells from at least some individuals early after treatment. This occurs through changes in PLAT promoter activity by steroid treatment in some donors. The variability in response of PLAT to steroids may underlie steroid responsiveness in patients.
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