title: SFRP1 promoter methylation and expression in human trabecular meshwork cells.
abstract: Glaucoma is a leading cause of blindness worldwide. In primary open angle glaucoma (POAG) patients, impaired trabecular meshwork (TM) function results in elevated intraocular pressure (IOP), which is the primary risk factor of developing optic neuropathy. Our previous studies showed that Wnt signaling pathway components are expressed in the human TM (HTM), and the Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) is elevated in the glaucomatous TM (GTM). Elevated SFRP1 increased IOP in mice eyes and in perfusion cultured anterior segments of the human eye. However, the cause of elevated SFRP1 in the GTM remains unknown. Promoter methylation plays a key role in regulating SFRP1 expression in certain cancer cells. In light of this, we studied whether promoter methylation is also involved in SFRP1 differential expression in the TM. Two normal TM (NTM) and two GTM cell strains were cultured for an additional 7 days after they were confluent. RNA and genomic DNA (gDNA) were isolated simultaneously to compare SFRP1 expression levels by quantitative PCR (qPCR) and to determine SFRP1 promoter methylation status by bisulfite conversion and methylation-sensitive high resolution melting analysis (MS-HRM). To study whether DNA methylation inhibitors affect SFRP1 expression in TM cells, the four TM cell strains were treated with or without 2 μM 5-aza-2'-deoxycytidine (AZA-dC) for 4 days. RNA was isolated to compare SFRP1 expression by qPCR. In addition, a human cancer cell line, NCI-H460, was used as a positive control. We found that the two GTM cell strains had significantly higher expression levels of SFRP1 than the two NTM cell strains. However, the SFRP1 promoter of all four TM cell strains was unmethylated. In addition, AZA-dC treatment did not affect SFRP1 expression in any of the TM cell strains (n = 3, p > 0.05). In contrast, the hypermethylated SFRP1 promoter of NCI-H460 cells was partially demethylated by the same treatment. AZA-dC treatment also elevated SFRP1 expression by approximately two fold in NCI-H460 cells (n = 3, p < 0.01). Our data suggest that the differential expression of SFRP1 in HTM cells is not due to differential promoter methylation.
authors.affiliation: North Texas Eye Research Institute, Department of Cell Biology & Anatomy, University of North Texas Health Science Center, CBH440, Fort Worth, TX 76107, USA. weiming.mao@unthsc.edu
authors.forename: Weiming
authors.initials: W
authors.lastname: Mao
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Rubin
authors.forename: Nancy
authors.initials: N
authors.lastname: Anoruo
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wordinger
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002478:Cells, Cultured; D019175:DNA Methylation; D015254:DNA Modification Methylases; D017931:DNA Primers / Q000737:chemistry; D000077209:Decitabine; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D005902:Glaucoma, Open-Angle / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D011401:Promoter Regions, Genetic / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000378:metabolism*; D000094742:Secreted Frizzled-Related Proteins
chemical_list: D001374:Azacitidine; D015254:DNA Modification Methylases; D017931:DNA Primers; D000077209:Decitabine; D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins