Abstract
Purpose: :
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.
Methods: :
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 identified differentially expressed genes with statistical significance defined as a q-value less than 5%. TightClust cluster analysis grouped significant genes by temporal expression patterns. 50 clusters resulted. Clusters up regulated at only 12 hrs were evaluated by Metacore transcription factor network algorithms; the presence of pathway start or end nodes were determined. Clusters down regulated only at 12 hrs were analyzed similarly. Resulting networks were assessed for ECM regulation themes.
Results: :
Genes in clusters down regulated by TNFα at 12 hrs implicate the integrin α2β1/focal adhesion kinase 1 (FAK1) pathway. This involves carboxy terminal binding protein 1 (CtBP1), which works with APC to sequester β-catenin for ubiquitination. For TNFα up regulated genes at 12 hrs, the FAK1 pathway is not active; β-catenin can bind T-cell-specific transcription factor (TCF) and N-cadherin, enabling Wnt pathway activation and cell-cell interactions, respectively. The extracellular signal-regulated kinase 1 (ERK1) pathway increases the level of TGF-β, also stimulating the accumulation of β-catenin and TCF promoter activity. The p38 and c-Jun amino-terminal kinase (JNK) pathways contribute to the availability of β-catenin by recruiting axin and inhibiting glycogen synthase kinase, components of the β-catenin degradation pathway.
Conclusions: :
β-catenin appears to be a key convergence point, linking the canonical Wnt pathway, N-cadherin, several key ECM components, and contributing to intraocular pressure (IOP) regulation.
Keywords: extracellular matrix • trabecular meshwork • transcription factors