Abstract
PurposeElevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG) results from glaucomatous damage to the trabecular meshwork (TM). The glaucoma-associated factor TGFβ2 is increased in aqueous humor and TM of POAG patients. We hypothesize that histone acetylation has a role in dysregulated TGFβ2 expression.MethodsProtein acetylation was compared between nonglaucomatous TM (NTM) and glaucomatous TM (GTM) cells using Western immunoblotting (WB). Nonglaucomatous TM cells were treated with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor for 4 days. Total and nuclear proteins, RNA, and nuclear protein-DNA complexes were harvested for WB, quantitative PCR (qPCR), and chromatin immunoprecipitation (ChIP) assays, respectively. Paired bovine eyes were perfused with TDP-A versus DMSO, or TDP-A versus TDP-A plus the TGFβ pathway inhibitor LY364947 for 5 to 9 days. Intraocular pressure, TM, and perfusate proteins were compared.ResultsWe found increased acetylated histone 3 and total protein acetylation in the GTM cells and TDP-A treated NTM cells. Chromatin immunoprecipitation assays showed that TDP-A induced histone hyperacetylation associated with the TGFβ2 promoter. This change of acetylation significantly increased TGFβ2 mRNA and protein expression in NTM cells. In perfusion-cultured bovine eyes, TDP-A increased TGFβ2 in the perfusate as well as elevated IOP. Histologic and immunofluorescent analyses showed increased extracellular matrix and cytoskeletal proteins in the TM of TDP-A treated bovine eyes. Cotreatment with the TGFβ pathway inhibitor LY364947 blocked TDP-A–induced ocular hypertension.ConclusionsOur results suggest that histone acetylation has an important role in increased expression of the glaucoma-associated factor TGFβ2. Histone hyperacetylation may be the initiator of glaucomatous damage to the TM.
Highlights
Chromatin immunoprecipitation assays showed that TDPA induced histone hyperacetylation associated with the transforming growth factorb2 (TGFb2) promoter
Our results suggest that histone acetylation has an important role in increased expression of the glaucoma-associated factor TGFb2
Three nonglaucomatous TM (NTM) and three glaucomatous TM (GTM) cell strains were cultured to confluency, and whole cell lysate and nuclear protein were harvested for Western immunoblotting (WB)
Summary
Protein acetylation was compared between nonglaucomatous TM (NTM) and glaucomatous TM (GTM) cells using Western immunoblotting (WB). Nonglaucomatous TM cells were treated with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor for 4 days. Paired bovine eyes were perfused with TDP-A versus DMSO, or TDP-A versus TDP-A plus the TGFb pathway inhibitor LY364947 for 5 to 9 days. TM cells migrated from TM explants onto culture plates. Nonglaucomatous TM and GTM cells were cultured in Dulbecco’s modified Eagle’s low glucose medium (DMEM; Sigma-Aldrich Corp., St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Atlas Biologicals, Collins, CO, USA), 1% penicillin þ streptomycin (Sigma-Aldrich Corp.), and 2 mM Lglutamine (GE Healthcare Life Sciences, Logan, UT, USA)
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