Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells

Allan R. Shepard, Nasreen Jacobson, John H. Fingert, Edwin M. Stone, Val C. Sheffield, Abbot F. Clark

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

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 was quantified by Western immunoblot analysis of protein levels and quantitative 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 [EC 50], ∼ 10 nM) and reversal by the glucocorticoid antagonist, RU486, implicates the glucocorticoid receptor (GR). In an interesting observation, RU486 alone acted as a partial agonist to MYOC expression. Treatment of TM cells with the protein synthesis inhibitor cycloheximide abolished the Dex induction, suggesting an indirect effect of the GR on MYOC expression. In addition, the RNA synthesis inhibitor actinomycin D also blocked Dex induction, indicating that the Dex effect was due to increased MYOC transcription. Analysis of up to 2700 nucleotides (nt) of the MYOC gene 5′-flanking region in luciferase reporter constructs showed no Dex induction, despite the presence of multiple putative glucocorticoid response element (GRE)-like 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. Characterization of the transcription factors that mediate the secondary response will shed new light on the pathophysiology of steroid-induced ocular hypertension and glaucoma.

Original languageEnglish
Pages (from-to)3173-3181
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume42
Issue number13
StatePublished - 1 Dec 2001

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