Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells

PURPOSE. To characterize the glucocorticoid responsiveness of the glaucoma gene MYOC (myocilin/TIGR) in cultured human trabecular meshwork (TM) cells. METHODS. MYOC expression in two independently derived human TM cell lines w as quantified by Western immunoblot analysis of protein levels and quantita tive PCR analysis of mRNA levels. Promoter activity was measured indirectly with the luciferase reporter gene in a dual luciferase reporter assay, RESULTs. Application of the synthetic glucocorticoid dexamethasone (Dex) to Cultured TM cells at 100 nM resulted in a delayed (8 - 16 hours) induction of myocilin. The concentration dependence (median effective concentration [EC50], similar to 10 nM) and reversal by the glucocorticoid antagonist, RU 486, implicates the glucocorticoid receptor (GR). In an interesting observa tion, RU486 alone acted as a partial agonist to MYOC expression. Treatment of TM cells with the protein synthesis inhibitor cycloheximide abolished th e Dex induction, suggesting an indirect effect of the GR on MYOC expression . In addition, the RNA synthesis inhibitor actinomycin D also blocked Dex i nduction, indicating that the Dex effect was due to increased MYOC transcri ption. Analysis of up to 2700 nucleotides (nt) of the MYOC gene 5 ' -flanki ng region in luciferase reporter constructs showed no Dex induction, despit e the presence of multiple putative glucocorticoid response element (GRE)-l ike half-sites in the MYOC promoter and the presence of an intact cellular GR-mediated signaling system. CONCLUSIONS. MYOC is a delayed secondary glucocorticoid-responsive gene. Ch aracterization of the transcription factors that mediate the secondary resp onse will shed new light on the pathophysiology of steroid-induced Ocular h ypertension and glaucoma.