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J.E. Donello, R. Grewal, D. Gil, P. Wong; Clusterin Upregulation by Brimondine . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2273.
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Purpose: The alpha 2-adrenergic receptor agonist Brimonidine protects the retina against various insults including ischemia, optic nerve crush, and light-induced damage. We have previously shown that there are gene expression changes associated with Brimonidine administration using micro-array analysis. One of the observations made from that study was that Brimonidine treatment affects clusterin mRNA expression. Clusterin has been implicated to play a role in retinal degeneration and has recently been found to be a main component of drusen. In the current communication we have utilised a cell culture model system to elucidate the receptor subtype (2a, 2b, or 2c) involved in the brimonidine regulated modulation of clusterin gene expression. Methods: An adenovirus-mediated transfection was used to express the three human receptor subtypes individually in PC12 and HEK-293 cells. Cells were cultured in DMEM and treated with 100 nM of Brimondine for 0,3,6,12 and 24 hours before isolation and RNA extraction. Total RNA was isolated and used to make Northern blots, which were probed with a clusterin radiolabelled cDNA probe. Results: Untreated PC12 cells expressing the receptor subtypes, and wildtype PC12 cells show a steady level of clusterin expression. Preliminary results suggest that that there is an induction of clusterin mRNA levels 12 hours after Brimonidine treatment in PC12 cells expressing the 2b-adrenergic receptor subtype. Cells expressing the 2a or 2c receptor do not show this induction. HEK-293 cells expressing the 2b receptor do not show an induction of clusterin expression, however cells expressing the 2a receptor show an induction of clusterin expression 24 hours after Brimonidine treatment. Conclusions: Initial experiments suggest that Brimondine up-regulates clusterin mRNA levels, and this is done through the 2b-adrenergic receptor subtype in neuronal like cells. Initial results also suggest that the receptor subtypes elicit different responses in PC12 and HEK cell types. Further analysis of Brimonidine induced gene expression changes may help shed light on the mechanism by which Brimonidine protects the retina.
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