Htm Cell Differentiation and Gene Expression in the Gc-induced Steroid Glaucoma Model

Abstract

Abstract: : Purpose:To explore the role of morphological differentiation in the HTM cell model for glaucoma, with regards to the corticosteroid regulation of potentially important gene products using real time PCR. The study of gene expression in differentiated vs. non-differentiated HTM cells treated with dexamethasone (DEX) previously allowed us to identify TIGR/MYOC (myocilin) as a candidate gene for glaucoma; the current studies extend the evaluations to certain acute phase response (APR) and potential Alzheimer-related products under investigation. Methods:Gene expression was evaluated in HTM cells and skin fibroblasts at varying times after plating, at different stages of apparent morphological differentiation in culture using gene-specific probes for the real time PCR studies. Results: Real time PCR evaluations of GC treatments to morphologically differentiated HTM cells with and without growth factor (bFGF) treatments confirmed a major gene regulation for certain APR/Alzheimer-related gene products previously identified in a collaborative study using microarray methods with Vollrath’s group at Stanford (see Zimmerman et al., ARVO 2002). The GC up-regulation of AACT (alpha-1 antichymotrypsin), APOD(apolipoprotein D), SLP1(serum secretory leukoproteinase inhibitor)and SAA1 (serum amyloid A precursor protein) as well as TIGR/MYOC was found to be substantially greater in differentiated HTM cells, with a much reduced to minimal regulation observed in partially-differentiated (late log phase to early confluency) HTM cells. Partially differentiated or differentiated skin fibroblasts showed a lack of the major GC effect, supporting the cell type specificity of the HTM cell gene regulation. Conclusions: The finding that prolonged DEX treatment of differentiated but not undifferentiated HTM cells or skin fibroblasts results in the substantial induction of mRNA for certain APR/ Alzheimer's disease-associated genes supports an interest in their potential role(s) in glaucoma. Further evaluations of cellular stress such as oxidative injury regarding perturbations in the genomic and proteomic profiles of differentiated HTM cells, as well as studies of relevant gene expression in glaucomatous tissue are planned; these may help reveal useful information concerning homeostatic function vs. glaucoma pathogenic mechanisms in the trabecular meshwork.

Keywords: gene/expression • trabecular meshwork • molecular biology