title: Analysis of glucocorticoid-induced MYOC expression in human trabecular meshwork cells.
abstract: To understand the regulatory mechanisms governing glucocorticoid-mediated MYOC induction in human trabecular meshwork (HTM) cells, the expression and degradation of MYOC mRNA were quantified in HTM cells by Northern blot analysis, and the transcriptional activity of constructs containing variable lengths of putative MYOC promoters was assessed by luciferase reporter assay. Here, we confirmed that MYOC is a delayed secondary glucocorticoid-responsive gene by demonstrating that its transcription was not initiated immediately by the addition of dexamethasone (DEX) and was completely inhibited by treatment with cycloheximide. In addition, we demonstrated that MYOC mRNA is degraded very slowly, with approximately half persisting for at least 4 days, suggesting that its mRNA is intrinsically quite stable. Promoter analysis of up to 5271 base pairs upstream of MYOC revealed that luciferase induction by DEX was increased by 280 ± 34% in HTM cells. Moreover, DEX induction required the region between base pairs -2548 and -1541. However, the putative regulatory element exhibited little activity in other cell lines, including TM-5, 293A, SH-SY5Y, and human retinal pigment epithelium (RPE) cells. To our knowledge, this study provides the first evidence for the presence of a cis-acting region for secondary glucocorticoid responsiveness in the 5'-flanking sequences of MYOC. It will be a major step towards understanding the expression pattern of MYOC in HTM cells and TM tissue.
authors.affiliation: Center for Clinical Research, Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, South Korea.
authors.forename: Myung Kuk
authors.initials: MK
authors.lastname: Joe
authors.forename: Seongsoo
authors.initials: S
authors.lastname: Sohn
authors.forename: Tae Eun
authors.initials: TE
authors.lastname: Kim
authors.forename: Ji-eun
authors.initials: JE
authors.lastname: Im
authors.forename: Young Ran
authors.initials: YR
authors.lastname: Choi
authors.forename: Changwon
authors.initials: C
authors.lastname: Kee
mesh_terms: D015152:Blotting, Northern; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005938:Glucocorticoids / Q000494:pharmacology*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism*; D000099179:Myocilin
chemical_list: D003513:Cycloheximide; D003598:Cytoskeletal Proteins; D003907:Dexamethasone; D005136:Eye Proteins; D005938:Glucocorticoids; D006023:Glycoproteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D000099179:Myocilin