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Lauren Hayashi, Dongseok Choi, Kathryn Carr, Mary Kelley, Ted Acott; Transcriptional Co-regulatory Patterns Associated with TNFα Treatment of Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3551.
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© ARVO (1962-2015); The Authors (2016-present)
TNFα is a key mediator of therapeutic effects of laser trabeculoplasty on glaucoma. In trabecular meshwork (TM) cells, matrix metalloproteinases initiate extracellular matrix (ECM) turnover in response to TNFα. To further understand this remodeling and its effects on aqueous humor outflow resistance, studies were conducted to identify transcription factor binding sites and regulatory pathways involved in TM gene expression patterns after TNFα treatment.
Primary porcine TM cells were treated with recombinant human TNFα (10ng/ml). Purified RNA was collected for gene expression profiling after 12, 24, and 48 hrs. After normalizing, Significance Analysis of Microarrays was used to identify differentially expressed genes with statistical significance defined as a q-value of less than 5%. TightClust cluster analysis was used to group significant genes by temporal expression patterns. Fifty clusters were identified. Clusters up regulated at 24 and 48 hrs were evaluated using Metacore transcription factor network algorithms and the presence of pathway start or end nodes were determined. Clusters down regulated at 24 and 48 hrs were analyzed similarly. All resulting networks were assessed for ECM regulation themes.
Genes in clusters down regulated by TNFα at 24 hrs implicate the canonical SMAD and non-canonical ERK TGF-β pathway, Wnt pathway and versican transcription. For TNFα treated clusters up regulated at 24 hrs, it appears carboxy terminal binding protein 1 (CtBP1) and APC sequester β-catenin, produced via JNK and c-Jun, mark it for ubiquitination and repress the Wnt pathway. TGF-β is up regulated through the non-canonical JNK pathway and could bind CD44-anchored SMAD2 or -3. CD44 may also bind hyluronan, strengthening the cellular response to BMP-7. By 48 hrs BMP-7 is up regulated, antagonizing TGF-β, while CtBP still prevents β-catenin from activating the Wnt pathway. Meanwhile versican, Wnt, thrombospondin 1, fibronectin and fibrillin are associated with genes down regulated at 48 hrs after TNFα treatment.
Wnt and TGF-β contribute to ECM components and intraocular pressure (IOP) regulation. Previously we showed Wnt is active at 12 hrs (Hayashi, et al. ARVO 2012; 3250/A95.). By 24 hrs Wnt and some TGF-β pathways are deactivated. Full repression of TGF-β occurs by 48 hrs. Wnt is an early response mechanism while TGF-β has longer term effects on the ECM.
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