Abstract
Purpose: :
Long term dexamethasone (DEX) treatment is known to induce myocilin (MYOC) transcription in trabecular meshwork (TM) cells. Excess myocilin protein has been observed in the TM tissue in about 50% of primary open-angle glaucoma (POAG) cases (Lütjen-Drecoll et al. IOVS 1998;39:517). Accumulation of myocilin in the TM is thought to increase aqueous humor outflow resistance, causing elevation of intraocular pressure (IOP) (Tamm, ER. Prog Ret Eye Res 2002;21:395). Shepard and colleagues have previously shown that the translation inhibitor cycloheximide prevents the induction of MYOC by DEX in TM cells (IOVS 2001;42:3173). This implies the existence of an intermediate protein, which we refer to as the Inducer of MYOC Transcription (IMT), which is necessary for upregulation of MYOC transcription. The purpose of this project was to identify the time period during which the putative IMT protein is synthesized.
Methods: :
Primary culture human TM cells were grown to 75% confluence and treated with DEX as previously reported (Rozsa et al. Mol Vis 2006;12:125). DEX-treated and untreated cells were harvested at 0, 2, 4, 6, 10, 12, 18, 24, and 48 h. Total RNA was extracted using TRIzol® Reagent (Life Technologies) and reverse transcribed; MYOC transcript was quantified by TaqMan® assay (Applied Biosystems, Inc.). Presence of secreted myocilin in the spent media was monitored by Western blot using a polyclonal antibody against myocilin (Santa Cruz Biotechnology).
Results: :
We analyzed MYOC expression in three primary TM cell cultures by TaqMan® assay and normalized the data to β-actin expression levels. We observed that DEX treatment led to a small but detectable increase in MYOC transcription after 6 to10 h with an 8- to 14-fold increase by 48 hours. Western blot analysis of the spent media from one cell line showed increased expression of myocilin starting around 10 h after DEX induction and becoming more prominent at 18 h and later.
Conclusions: :
We find that induction of MYOC transcription begins within 6 to 10 h of exposure to DEX, implying that the putative IMT protein required for DEX induction of MYOC is expressed prior to this time frame. Analysis of the transcriptome and proteome of cells captured before this time will assist in the identification of the IMT protein, which will lead to a better understanding of the MYOC induction pathway as well as a potential new drug target for glaucoma treatment.
Keywords: gene/expression • trabecular meshwork • genetics