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1 From the Laboratory of Molecular and Developmental Biology and the 2 Laboratory of Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland; the 3 Duke University Eye Center, Durham, North Carolina; and the 4 Mayo Clinic, Rochester, Minnesota.
PURPOSE. To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM).
METHODS. mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-ß1 (1 ng/ml), dexamethasone (10-7 M), and mechanical stretch (10%).
RESULTS. mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 µg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 µg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-ß1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-ß1, this effect was observed much earlier (8–24 hours) after treatment.
CONCLUSIONS. Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.
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